48 results on '"Nicholas Tsakmaklis"'
Search Results
2. Natural history of Waldenström macroglobulinemia following acquired resistance to ibrutinib monotherapy
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Joshua N. Gustine, Shayna Sarosiek, Catherine A. Flynn, Kirsten Meid, Carly Leventoff, Timothy White, Maria Luisa Guerrera, Lian Xu, Amanda Kofides, Nicholas Tsakmaklis, Manit Munshi, Maria Demos, Christopher J. Patterson, Xia Liu, Guang Yang, Zachary R. Hunter, Andrew R. Branagan, Steven P. Treon, and Jorge J. Castillo
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Diseases of the blood and blood-forming organs ,RC633-647.5 - Abstract
Ibrutinib is highly active and produces long-term responses in patients with Waldenström macroglobulinemia (WM), but acquired resistance can occur with prolonged treatment. We therefore evaluated the natural history and treatment outcomes in 51 WM patients with acquired resistance to ibrutinib monotherapy. The median time between ibrutinib initiation and discontinuation was 2 years (range, 0.4-6.5 years). Following discontinuation of ibrutinib, a rapid increase in serum immunoglobulin M level was observed in 60% (29/48) of evaluable patients, of whom ten acutely developed symptomatic hyperviscosity. Forty-eight patients (94%) received salvage therapy after ibrutinib. The median time to salvage therapy after ibrutinib cessation was 18 days (95% confidence interval [CI]: 13-27). The overall and major response rates to salvage therapy were 56% and 44%, respectively, and the median duration of response was 48 months (95% CI: 34-not reached). Quadruple-class (rituximab, alkylator, proteasome inhibitor, ibrutinib) exposed disease (odds ratio [OR] 0.20, 95% CI: 0.05-0.73) and salvage therapy ≤7 days after discontinuing ibrutinib (OR 4.12, 95% CI: 1.07- 18.9) were identified as independent predictors of a response to salvage therapy. The 5-year overall survival (OS) following discontinuation of ibrutinib was 44% (95% CI: 26-75). Response to salvage therapy was associated with better OS after ibrutinib (hazard ratio 0.08, 95% CI: 0.02-0.38). TP53 mutations were associated with shorter OS, while acquired BTK C481S mutations had no impact. Our findings reveal that continuation of ibrutinib until subsequent treatment is associated with improved disease control and clinical outcomes.
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- 2021
- Full Text
- View/download PDF
3. CXCR4 S338X clonality is an important determinant of ibrutinib outcomes in patients with Waldenström macroglobulinemia
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Joshua N. Gustine, Lian Xu, Nicholas Tsakmaklis, Maria G. Demos, Amanda Kofides, Jiaji G. Chen, Xia Liu, Manit Munshi, Maria Luisa Guerrera, Gloria G. Chan, Christopher J. Patterson, Andrew Keezer, Kirsten Meid, Toni Dubeau, Guang Yang, Zachary R. Hunter, Steven P. Treon, and Jorge J. Castillo
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Specialties of internal medicine ,RC581-951 - Published
- 2019
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4. Response and Survival Outcomes to Ibrutinib Monotherapy for Patients With Waldenström Macroglobulinemia on and off Clinical Trials
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Jorge J. Castillo, Joshua N. Gustine, Kirsten Meid, Catherine A. Flynn, Maria G. Demos, Maria L. Guerrera, Cristina Jimenez, Amanda Kofides, Xia Liu, Manit Munshi, Nicholas Tsakmaklis, Christopher J. Patterson, Lian Xu, Guang Yang, Zachary R. Hunter, and Steven P. Treon
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Diseases of the blood and blood-forming organs ,RC633-647.5 - Abstract
Abstract. Ibrutinib is the first approved therapy for symptomatic patients with Waldenström macroglobulinemia (WM). The approval was based on a single, multicenter, phase II trial in previously treated WM patients. We sought to evaluate whether there were differences in clinical characteristics, response, and survival outcomes to ibrutinib monotherapy between WM patients treated on and off clinical trials. Treatment naïve and previously treated patients who received ibrutinib monotherapy at our institution and participated in two prospective studies (ON trial; n = 72) or a prospective database (OFF trial; n = 157) were included. The median times from WM diagnosis to ibrutinib initiation were 3.1 and 3.5 years for ON and OFF trial patients, respectively (p = 0.38). Similar rates of categorical response at 6, 12, and 24 months and at best response were also observed between ON trial and OFF trial patients. The 4-year PFS and OS rates for ON trial and OFF trial patients were 72% and 63%, respectively (log-rank p = 0.14) and 83% and 81%, respectively (log-rank p = 0.14). CXCR4 mutations impacted response and survival outcomes to ibrutinib monotherapy. The 4-year rates of ibrutinib discontinuation in ON and OFF trial patients were 36% and 44%, respectively (p = 0.11). Ibrutinib is effective in the routine clinical care of both treatment-naïve and previously treated WM patients. The findings of our study validate the efficacy of ibrutinib monotherapy reported in multiple phase II clinical trials.
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- 2020
- Full Text
- View/download PDF
5. Response and survival predictors in a cohort of 319 patients with Waldenström macroglobulinemia treated with ibrutinib monotherapy
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Jorge J. Castillo, Shayna R. Sarosiek, Joshua N. Gustine, Catherine A. Flynn, Carly R. Leventoff, Timothy P. White, Kirsten Meid, Maria L. Guerrera, Amanda Kofides, Xia Liu, Manit Munshi, Nicholas Tsakmaklis, Zachary R. Hunter, Christopher J. Patterson, Andrew R. Branagan, and Steven P. Treon
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Pyrimidines ,Piperidines ,Adenine ,Humans ,Pyrazoles ,Hematology ,Waldenstrom Macroglobulinemia ,Aged - Abstract
Bruton tyrosine kinase (BTK) inhibitors are the only FDA-approved treatments for Waldenström macroglobulinemia (WM). Factors prognostic of survival and predictive of response to BTK inhibitors remained to be clarified. We evaluated 319 patients with WM to identify predictive and prognostic factors on ibrutinib monotherapy. Logistic and Cox proportional-hazard regression models were fitted for response and survival. Multiple imputation analyses were used to address bias associated with missing data. Major (partial response or better) and deep responses (very good partial response or better) were attained in 78% and 28% of patients. CXCR4 mutations were associated with lower odds of major (odds ratio [OR], 0.2; 95% confidence interval [CI], 0.1-0.5; P < .001) and deep response (OR, 0.3; 95% CI, 0.2-0.6; P = .001). CXCR4 mutations (hazard ratio [HR], 2.0; 95% CI, 1.2-3.4; P = .01) and platelet count 100 K/uL or less (HR, 2.5; 95% CI, 1.3-4.9; P = .007) were associated with worse progression-free survival (PFS). We proposed a scoring system using these 2 factors. The median PFS for patients with 0, 1, and 2 risk factors were not reached, 5 years and 3 years (P < .001). Patients with 2 risk factors had HR 2.2 (95% CI, 1.3-3.8; P = .004) compared with 1 factor, and patients with 1 factor had HR 2.3 (95% CI, 1.1-5.1; P = .03) compared with 0 factors. Age ≥65 years was the only factor associated with overall survival (HR, 3.2; 95% CI, 1.4-7.0; P = .005). Multiple imputation analyses did not alter our results. Our study confirms the predictive and prognostic value of CXCR4 mutations in patients with WM treated with ibrutinib monotherapy.
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- 2022
6. Venetoclax in Previously Treated Waldenström Macroglobulinemia
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Christopher J. Patterson, Lian Xu, Tanya Siddiqi, Maria Luisa Guerrera, Shayna Sarosiek, Zachary R. Hunter, Andrew R. Branagan, Xia Liu, Carly Leventoff, Catherine A Flynn, Ranjana H. Advani, Kirsten Meid, Nicholas Tsakmaklis, Maria Demos, Matthew S. Davids, Guang Yang, Amanda Kofides, Timothy P White, Manit Munshi, Steven P. Treon, Jorge J. Castillo, Richard R. Furman, and John N. Allan
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Adult ,Male ,Cancer Research ,Receptors, CXCR4 ,Time Factors ,Antineoplastic Agents ,chemistry.chemical_compound ,Biomarkers, Tumor ,Medicine ,Humans ,Prospective Studies ,BCL2 ANTAGONIST ,Aged ,Aged, 80 and over ,Sulfonamides ,Venetoclax ,business.industry ,Waldenstrom macroglobulinemia ,Middle Aged ,medicine.disease ,Bridged Bicyclo Compounds, Heterocyclic ,Progression-Free Survival ,United States ,Oncology ,chemistry ,Proto-Oncogene Proteins c-bcl-2 ,Mutation ,Myeloid Differentiation Factor 88 ,Cancer research ,Disease Progression ,Female ,Waldenstrom Macroglobulinemia ,Previously treated ,business - Abstract
PURPOSE BCL2 is overexpressed and confers prosurvival signaling in malignant lymphoplasmacytic cells in Waldenström macroglobulinemia (WM). Venetoclax is a potent BCL2 antagonist and triggers in vitro apoptosis of WM cells. The activity of venetoclax in WM remains to be clarified. PATIENTS AND METHODS We performed a multicenter, prospective phase II study of venetoclax in patients with previously treated WM ( NCT02677324 ). Venetoclax was dose-escalated from 200 mg to a maximum dose of 800 mg daily for up to 2 years. RESULTS Thirty-two patients were evaluable, including 16 previously exposed to Bruton tyrosine kinase inhibitors (BTKis). All patients were MYD88 L265P–mutated, and 17 carried CXCR4 mutations. The median time to minor and major responses was 1.9 and 5.1 months, respectively. Previous exposure to BTKis was associated with a longer time to response (4.5 v 1.4 months; P < .001). The overall, major, and very good partial response rates were 84%, 81%, and 19%, respectively. The major response rate was lower in those with refractory versus relapsed disease (50% v 95%; P = .007). The median follow-up time was 33 months, and the median progression-free survival was 30 months. CXCR4 mutations did not affect treatment response or progression-free survival. The only recurring grade ≥ 3 treatment-related adverse event was neutropenia (n = 14; 45%), including one episode of febrile neutropenia. Laboratory tumor lysis without clinical sequelae occurred in one patient. No deaths have occurred. CONCLUSION Venetoclax is safe and highly active in patients with previously treated WM, including those who previously received BTKis. CXCR4 mutation status did not affect treatment response.
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- 2023
7. The HCK/BTK inhibitor KIN-8194 is active in MYD88-driven lymphomas and overcomes mutated BTKCys481 ibrutinib resistance
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Amanda Kofides, Kirsten Meid, Sara J. Buhrlage, Jinhua Wang, Manit Munshi, Jorge J. Castillo, Jiaji G. Chen, Kenneth C. Anderson, Michael D. Cameron, Guang Yang, Maria Luisa Guerrera, Xia Liu, Lian Xu, Nicholas Tsakmaklis, Nikhil C. Munshi, Jianwei Che, Li Tan, Maria Demos, Nathanael S. Gray, Christopher J. Patterson, Steven P. Treon, and Zachary R. Hunter
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Proto-Oncogene Proteins c-hck ,Lymphoma ,Immunology ,Antineoplastic Agents ,Mice, SCID ,Biochemistry ,chemistry.chemical_compound ,Piperidines ,Pharmacokinetics ,Mice, Inbred NOD ,Cell Line, Tumor ,hemic and lymphatic diseases ,Agammaglobulinaemia Tyrosine Kinase ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,Bruton's tyrosine kinase ,Protein Kinase Inhibitors ,Lymphoid Neoplasia ,biology ,Venetoclax ,business.industry ,Adenine ,Waldenstrom macroglobulinemia ,Cell Biology ,Hematology ,medicine.disease ,In vitro ,chemistry ,Drug Resistance, Neoplasm ,Ibrutinib ,Mutation ,Myeloid Differentiation Factor 88 ,biology.protein ,Cancer research ,Female ,business - Abstract
Activating mutations in MYD88 promote malignant cell growth and survival through hematopoietic cell kinase (HCK)–mediated activation of Bruton tyrosine kinase (BTK). Ibrutinib binds to BTKCys481 and is active in B-cell malignancies driven by mutated MYD88. Mutations in BTKCys481, particularly BTKCys481Ser, are common in patients with acquired ibrutinib resistance. We therefore performed an extensive medicinal chemistry campaign and identified KIN-8194 as a novel dual inhibitor of HCK and BTK. KIN-8194 showed potent and selective in vitro killing of MYD88-mutated lymphoma cells, including ibrutinib-resistant BTKCys481Ser-expressing cells. KIN-8194 demonstrated excellent bioavailability and pharmacokinetic parameters, with good tolerance in rodent models at pharmacologically achievable and active doses. Pharmacodynamic studies showed sustained inhibition of HCK and BTK for 24 hours after single oral administration of KIN-8194 in an MYD88-mutated TMD-8 activated B-cell diffuse large B-cell lymphoma (ABC DLBCL) and BCWM.1 Waldenström macroglobulinemia (WM) xenografted mice with wild-type BTK (BTKWT)– or BTKCys481Ser-expressing tumors. KIN-8194 showed superior survival benefit over ibrutinib in both BTKWT- and BTKCys481Ser-expressing TMD-8 DLBCL xenografted mice, including sustained complete responses of >12 weeks off treatment in mice with BTKWT-expressing TMD-8 tumors. The BCL_2 inhibitor venetoclax enhanced the antitumor activity of KIN-8194 in BTKWT- and BTKCys481Ser-expressing MYD88-mutated lymphoma cells and markedly reduced tumor growth and prolonged survival in mice with BTKCys481Ser-expressing TMD-8 tumors treated with both drugs. The findings highlight the feasibility of targeting HCK, a key driver of mutated MYD88 pro-survival signaling, and provide a framework for the advancement of KIN-8194 for human studies in B-cell malignancies driven by HCK and BTK.
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- 2021
8. Phase 1 study of ibrutinib and the CXCR4 antagonist ulocuplumab in CXCR4-mutated Waldenström macroglobulinemia
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Nicholas Tsakmaklis, Aldo M. Roccaro, Timothy P White, Christopher J. Patterson, Guang Yang, Antonio Sacco, Xia Liu, Yang Cao, Zachary R. Hunter, Shayna Sarosiek, Carly Leventoff, Irene M. Ghobrial, Manit Munshi, Jorge J. Castillo, Lian Xu, Steven P. Treon, Catherine A Flynn, Kirsten Meid, Andrew R. Branagan, Amanda Kofides, Maria Luisa Guerrera, and Maria Demos
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Adult ,Receptors, CXCR4 ,medicine.medical_specialty ,Clinical Trials and Observations ,Immunology ,Ulocuplumab ,Antineoplastic Agents ,Antibodies, Monoclonal, Humanized ,Biochemistry ,CXCR4 ,Gastroenterology ,chemistry.chemical_compound ,Piperidines ,Internal medicine ,Humans ,Medicine ,Adverse effect ,Protein Kinase Inhibitors ,Aged ,CXCR4 antagonist ,biology ,business.industry ,Adenine ,Waldenstrom macroglobulinemia ,Cell Biology ,Hematology ,Middle Aged ,medicine.disease ,Rash ,chemistry ,Immunoglobulin M ,Ibrutinib ,Mutation ,biology.protein ,Waldenstrom Macroglobulinemia ,medicine.symptom ,business - Abstract
MYD88 and CXCR4 mutations are common in Waldenström macroglobulinemia (WM). Mutated CXCR4 (CXCR4Mut) impacts BTK-inhibitor response. We conducted a phase 1 trial of the CXCR4-antagonist ulocuplumab with ibrutinib in this first-ever study to target CXCR4Mut in WM. Ibrutinib was initiated at 420 mg/d with cycle 1 and continued until intolerance or progression; ulocuplumab was given cycles 1 to 6, with a 3 + 3 dose-escalation design. Each cycle was 4 weeks. Thirteen symptomatic patients, of whom 9 were treatment-naive patients were enrolled. Twelve were evaluable for response. At best response, their median serum immunoglobulin M declined from 5574 to 1114 mg/dL; bone marrow disease decreased from 65% to 10%, and hemoglobin increased from 10.1 to 14.2 g/dL (P < .001). The major and VGPR response rates were 100% and 33%, respectively, with VGPRs observed at lower ulocuplumab dose cohorts. Median times to minor and major responses were 0.9 and 1.2 months, respectively. With a median follow-up of 22.4 months, the estimated 2-year progression-free survival was 90%. The most frequent recurring grade ≥2 adverse events included reversible thrombocytopenia, rash, and skin infections. Ulocuplumab dose-escalation did not impact adverse events. The study demonstrates the feasibility of combining a CXCR4-antagonist with ibrutinib and provides support for the development of CXCR4-antagonists for CXCR4Mut WM. This trial was registered at www.clinicaltrials.gov as #NCT03225716.
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- 2021
9. Ibrutinib and Venetoclax in Previously Untreated Waldenström Macroglobulinemia
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Jorge J. Castillo, Shayna Sarosiek, Andrew R. Branagan, David J. Sermer, Catherine A. Flynn, Carly Leventoff, Megan Little, Timothy P White, Kirsten Meid, Alexa Canning, Maria Luisa Guerrera, Amanda Kofides, Xia Liu, Manit Munshi, Nicholas Tsakmaklis, Christopher J Patterson, Zachary R Hunter, and Steven P Treon
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
10. Bone marrow involvement and subclonal diversity impairs detection of mutated CXCR4 by diagnostic next‐generation sequencing in Waldenström macroglobulinaemia
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Maria Luisa Guerrera, Manit Munshi, Christopher J. Patterson, Kirsten Meid, Xia Liu, Joshua Gustine, Lian Xu, Jorge J. Castillo, Shayna Sarosiek, Guang Yang, Amanda Kofides, Andrew R. Branagan, Zachary R. Hunter, Nicholas Tsakmaklis, Maria Demos, and Steven P. Treon
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Receptors, CXCR4 ,medicine.disease_cause ,CXCR4 ,Article ,DNA sequencing ,law.invention ,03 medical and health sciences ,symbols.namesake ,chemistry.chemical_compound ,0302 clinical medicine ,Bone Marrow ,law ,Humans ,Point Mutation ,Medicine ,Polymerase chain reaction ,Sanger sequencing ,Mutation ,business.industry ,High-Throughput Nucleotide Sequencing ,Waldenstrom macroglobulinemia ,Hematology ,medicine.disease ,medicine.anatomical_structure ,chemistry ,030220 oncology & carcinogenesis ,Ibrutinib ,Cancer research ,symbols ,Bone marrow ,Waldenstrom Macroglobulinemia ,business ,030215 immunology - Abstract
CXCR4 mutations impact disease presentation and treatment outcomes in Waldenstrom macroglobulinaemia (WM). Non-uniform testing for CXCR4 mutations may account for discordant findings in WM clinical trials. We compared two approaches used in these trials for detection of the most common CXCR4 (S338X) variant: targeted next-generation sequencing (NGS) using unselected bone marrow (BM) samples, and combined allele-specific polymerase chain reaction (AS-PCR) and Sanger sequencing with unselected and CD19-selected BM samples. Our findings showed that targeted NGS frequently yielded false-negative results. Both CD19 selection and AS-PCR markedly improved detection of CXCR4S338X mutations. Sensitivity was adversely impacted by low BM involvement and CXCR4 mutation clonality.
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- 2021
11. Long-term follow-up of ibrutinib monotherapy in treatment-naive patients with Waldenstrom macroglobulinemia
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Noopur Raje, Elizabeth O'Donnell, Xia Liu, Kirsten Meid, Carly Leventoff, Zachary R. Hunter, Manit Munshi, Christopher J. Patterson, Amanda Kofides, Shayna Sarosiek, Steven P. Treon, Lian Xu, Andrew R. Branagan, Joshua Gustine, Andrew Yee, Guang Yang, Timothy P White, Maria Demos, Jorge J. Castillo, Maria Luisa Guerrera, Nicholas Tsakmaklis, and Catherine A Flynn
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Adult ,Male ,Cancer Research ,medicine.medical_specialty ,Phases of clinical research ,CXCR4 ,Gastroenterology ,Article ,chemistry.chemical_compound ,Hematoma ,Targeted therapies ,Piperidines ,Internal medicine ,medicine ,Humans ,Prospective Studies ,Adverse effect ,Aged ,Aged, 80 and over ,business.industry ,B-cell lymphoma ,Adenine ,Respiratory infection ,Waldenstrom macroglobulinemia ,Atrial fibrillation ,Hematology ,Middle Aged ,medicine.disease ,Prognosis ,Survival Rate ,Oncology ,chemistry ,Ibrutinib ,Female ,Waldenstrom Macroglobulinemia ,business ,Follow-Up Studies - Abstract
Herein, we present the final report of a single-center, prospective phase II study evaluating ibrutinib 420 mg once daily in 30 treatment-naive patients with Waldenstrom macroglobulinemia (WM). The present study is registered with ClinicalTrials.Gov (NCT02604511). With a median follow-up of 50 months, the overall, major, and VGPR response rates were 100%, 87%, and 30%. The VGPR rate was numerically but not significantly lower in patients with than without CXCR4 mutations (14% vs. 44%; p = 0.09). The median time to a minor response was 0.9 months, and to a major response was 1.9 months, though were longer in those with mutated CXCR4 at 1.7 months (p = 0.07) and 7.3 months (p = 0.01). Six patients had disease progression. The median progression-free survival (PFS) was not reached, and the 4-year PFS rate was 76%. There was also a non-significant lower 4-year PFS rate in patients with than without CXCR4 mutations (59% vs. 92%; p = 0.06). The most common treatment-related adverse events were fatigue, upper respiratory infection, and hematoma. Atrial fibrillation occurred in 20% of patients. Ibrutinib monotherapy induced durable responses in treatment-naive patients with WM. CXCR4 mutations impacted VGPR attainment, time to major response, and 4-year PFS rate.
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- 2021
12. CXCR4 S338X clonality is an important determinant of ibrutinib outcomes in patients with Waldenström macroglobulinemia
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Xia Liu, Gloria Chan, Manit Munshi, Nicholas Tsakmaklis, Kirsten Meid, Jorge J. Castillo, Jiaji G. Chen, Joshua Gustine, Guang Yang, Christopher J. Patterson, Andrew Keezer, Amanda Kofides, Maria Luisa Guerrera, Lian Xu, Toni Dubeau, Zachary R. Hunter, Steven P. Treon, and Maria Demos
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Oncology ,Very Good Partial Response ,medicine.medical_specialty ,business.industry ,Waldenstrom macroglobulinemia ,Hematology ,medicine.disease ,Stimulus Report ,CXCR4 ,chemistry.chemical_compound ,chemistry ,Internal medicine ,Ibrutinib ,medicine ,Biomarker (medicine) ,In patient ,business - Abstract
Key Points CXCR4 S338X clonality ≥25% is associated with lower very good partial response and shorter progression-free survival to ibrutinib. CXCR4 S338X clonality assessment represents a novel biomarker to predict outcomes to ibrutinib in Waldenström macroglobulinemia patients.
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- 2019
13. Natural history of Waldenström macroglobulinemia following acquired resistance to ibrutinib monotherapy
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Maria Luisa Guerrera, Christopher J. Patterson, Maria Demos, Carly Leventoff, Andrew R. Branagan, Amanda Kofides, Zachary R. Hunter, Joshua Gustine, Jorge J. Castillo, Nicholas Tsakmaklis, Xia Liu, Shayna Sarosiek, Timothy P White, Steven P. Treon, Catherine A Flynn, Manit Munshi, Lian Xu, Guang Yang, and Kirsten Meid
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Oncology ,medicine.medical_specialty ,business.industry ,Adenine ,Waldenstrom macroglobulinemia ,Salvage therapy ,Hematology ,medicine.disease ,Discontinuation ,Natural history ,chemistry.chemical_compound ,Acquired resistance ,Pyrimidines ,chemistry ,Piperidines ,Internal medicine ,Ibrutinib ,Proteasome inhibitor ,medicine ,Humans ,Pyrazoles ,Rituximab ,Waldenstrom Macroglobulinemia ,business ,medicine.drug - Abstract
Ibrutinib is highly active and produces long-term responses in patients with Waldenström macroglobulinemia (WM), but acquired resistance can occur with prolonged treatment. We therefore evaluated the natural history and treatment outcomes in 51 WM patients with acquired resistance to ibrutinib monotherapy. The median time between ibrutinib initiation and discontinuation was 2 years (range, 0.4-6.5 years). Following discontinuation of ibrutinib, a rapid increase in serum immunoglobulin M level was observed in 60% (29/48) of evaluable patients, of whom ten acutely developed symptomatic hyperviscosity. Forty-eight patients (94%) received salvage therapy after ibrutinib. The median time to salvage therapy after ibrutinib cessation was 18 days (95% confidence interval [CI]: 13-27). The overall and major response rates to salvage therapy were 56% and 44%, respectively, and the median duration of response was 48 months (95% CI: 34-not reached). Quadruple-class (rituximab, alkylator, proteasome inhibitor, ibrutinib) exposed disease (odds ratio [OR] 0.20, 95% CI: 0.05-0.73) and salvage therapy ≤7 days after discontinuing ibrutinib (OR 4.12, 95% CI: 1.07- 18.9) were identified as independent predictors of a response to salvage therapy. The 5-year overall survival (OS) following discontinuation of ibrutinib was 44% (95% CI: 26-75). Response to salvage therapy was associated with better OS after ibrutinib (hazard ratio 0.08, 95% CI: 0.02-0.38). TP53 mutations were associated with shorter OS, while acquired BTK C481S mutations had no impact. Our findings reveal that continuation of ibrutinib until subsequent treatment is associated with improved disease control and clinical outcomes.
- Published
- 2021
14. Cell‐free <scp>DNA</scp> analysis for detection of <scp> MYD88 L265P </scp> and <scp> CXCR4 S338X </scp> mutations in <scp>W</scp> aldenström macroglobulinemia
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Lian Xu, Catherine A Flynn, Maria Luisa Guerrera, Christopher J. Patterson, Jorge J. Castillo, Zachary R. Hunter, Guang Yang, Amanda Kofides, Timothy P White, Nicholas Tsakmaklis, Kirsten Meid, Steven P. Treon, Xia Liu, Shayna Sarosiek, Maria Demos, Carly Leventoff, Joshua Gustine, Andrew R. Branagan, and Manit Munshi
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Oncology ,medicine.medical_specialty ,business.industry ,Macroglobulinemia ,Waldenstrom macroglobulinemia ,Hematology ,Gold standard (test) ,medicine.disease ,CXCR4 ,law.invention ,03 medical and health sciences ,0302 clinical medicine ,medicine.anatomical_structure ,Cell-free fetal DNA ,law ,030220 oncology & carcinogenesis ,Internal medicine ,medicine ,Bone marrow ,business ,Genotyping ,Polymerase chain reaction ,030215 immunology - Abstract
MYD88L265P and CXCR4S338X variants are highly prevalent and impact disease presentation, prognosis, and/or treatment outcome in Waldenstrom's Macroglobulinemia (WM). The use of bone marrow (BM) aspirate represents the current "gold standard" for molecular testing in WM. Although these variants are identified in peripheral blood (PB), the diagnostic yield in PB is inferior to BM, particularly for previously treated patients. Tumor enrichment can significantly improve testing sensitivity, but is not feasible in most clinical laboratories. Recent studies have demonstrated the feasibility of identifying MYD88 and CXCR4 mutations using cell-free DNA (cfDNA) from the plasma of WM patients. We therefore prospectively collected matched BM and PB samples from 28 consecutive WM patients. Overall, five different tissue fractions were isolated for analysis: CD19-selected BM, unselected BM, CD19-selected PB, unselected PB, and cfDNA. Quantitative allele-specific polymerase chain reaction assays for MYD88L265P and CXCR4S338X mutations were performed for each tissue fraction, and findings benchmarked against CD19-selected BM. Both MYD88L265P and CXCR4S338X were identified with high sensitivity and specificity in cfDNA derived from the plasma of WM patients, including previously treated patients. The use of cfDNA represents a non-invasive, convenient, and potentially cost-effective method for genotyping patients with WM. This article is protected by copyright. All rights reserved.
- Published
- 2021
15. Cell-free DNA analysis for detection of MYD88
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Maria G, Demos, Zachary R, Hunter, Lian, Xu, Nicholas, Tsakmaklis, Amanda, Kofides, Manit, Munshi, Xia, Liu, Maria Luisa, Guerrera, Carly R, Leventoff, Timothy P, White, Catherine A, Flynn, Kirsten, Meid, Christopher J, Patterson, Guang, Yang, Andrew R, Branagan, Shayna, Sarosiek, Jorge J, Castillo, Steven P, Treon, and Joshua N, Gustine
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Receptors, CXCR4 ,DNA Mutational Analysis ,Myeloid Differentiation Factor 88 ,Humans ,Point Mutation ,Waldenstrom Macroglobulinemia - Published
- 2021
16. Response and Survival Outcomes to Ibrutinib Monotherapy for Patients With Waldenström Macroglobulinemia on and off Clinical Trials
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Amanda Kofides, Nicholas Tsakmaklis, Catherine A Flynn, Jorge J. Castillo, Maria Demos, Christopher J. Patterson, Steven P. Treon, Maria Luisa Guerrera, Xia Liu, Guang Yang, Lian Xu, Zachary R. Hunter, Joshua Gustine, Manit Munshi, Kirsten Meid, and Cristina Jimenez
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Oncology ,medicine.medical_specialty ,business.industry ,lcsh:RC633-647.5 ,Waldenstrom macroglobulinemia ,Phases of clinical research ,Hematology ,lcsh:Diseases of the blood and blood-forming organs ,medicine.disease ,Article ,Discontinuation ,Therapy naive ,Clinical trial ,chemistry.chemical_compound ,chemistry ,Ibrutinib ,Internal medicine ,medicine ,business ,Prospective cohort study ,Previously treated - Abstract
Ibrutinib is the first approved therapy for symptomatic patients with Waldenström macroglobulinemia (WM). The approval was based on a single, multicenter, phase II trial in previously treated WM patients. We sought to evaluate whether there were differences in clinical characteristics, response, and survival outcomes to ibrutinib monotherapy between WM patients treated on and off clinical trials. Treatment naïve and previously treated patients who received ibrutinib monotherapy at our institution and participated in two prospective studies (ON trial; n = 72) or a prospective database (OFF trial; n = 157) were included. The median times from WM diagnosis to ibrutinib initiation were 3.1 and 3.5 years for ON and OFF trial patients, respectively (p = 0.38). Similar rates of categorical response at 6, 12, and 24 months and at best response were also observed between ON trial and OFF trial patients. The 4-year PFS and OS rates for ON trial and OFF trial patients were 72% and 63%, respectively (log-rank p = 0.14) and 83% and 81%, respectively (log-rank p = 0.14). CXCR4 mutations impacted response and survival outcomes to ibrutinib monotherapy. The 4-year rates of ibrutinib discontinuation in ON and OFF trial patients were 36% and 44%, respectively (p = 0.11). Ibrutinib is effective in the routine clinical care of both treatment-naïve and previously treated WM patients. The findings of our study validate the efficacy of ibrutinib monotherapy reported in multiple phase II clinical trials.
- Published
- 2020
17. Ixazomib, dexamethasone, and rituximab in treatment-naive patients with Waldenström macroglobulinemia: long-term follow-up
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Steven P. Treon, Jorge J. Castillo, Xia Liu, Guang Yang, Catherine A Flynn, Christopher J. Patterson, Amanda Kofides, Kirsten Meid, Nicholas Tsakmaklis, Manit Munshi, Maria Demos, Jiaji Chen, Zachary R. Hunter, and Maria Luisa Guerrera
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Boron Compounds ,medicine.medical_specialty ,Clinical Trials and Observations ,Glycine ,Gastroenterology ,Dexamethasone ,Ixazomib ,chemistry.chemical_compound ,Internal medicine ,medicine ,Humans ,Prospective Studies ,Prospective cohort study ,Adverse effect ,Very Good Partial Response ,business.industry ,Waldenstrom macroglobulinemia ,Hematology ,medicine.disease ,Clinical trial ,chemistry ,Myeloid Differentiation Factor 88 ,Rituximab ,Waldenstrom Macroglobulinemia ,business ,medicine.drug ,Follow-Up Studies - Abstract
Proteasome inhibition is a standard of care for the primary treatment of patients with Waldenström macroglobulinemia (WM). We present the long-term follow-up of a prospective, phase II clinical trial that evaluated the combination of ixazomib, dexamethasone, and rituximab (IDR) in 26 treatment-naive patients with WM. IDR was administered as 6 monthly induction cycles followed by 6 every-2-month maintenance cycles. The MYD88 L265P mutation was detected in all patients, and CXCR4 mutations were detected in 15 patients (58%). The median time to response (TTR) and time to major response (TTMR) were 2 and 6 months, respectively. Patients with and without CXCR4 mutations had median TTR of 3 months and 1 month, respectively (P = .003), and median TTMR of 10 months and 3 months, respectively (P = .31). The overall, major, and very good partial response (VGPR) rates were 96%, 77%, and 19%, respectively. The rate of VGPR in patients with and without CXCR4 mutations were 7% and 36%, respectively (P = .06). The median progression-free survival (PFS) was 40 months, the median duration of response (DOR) was 38 months, and the median time to next treatment (TTNT) was 40 months. PFS, DOR, and TTNT were not affected by CXCR4 mutational status. The safety profile was excellent with no grade 4 adverse events or deaths to date. IDR provides a safe and effective frontline treatment option for symptomatic patients with WM. This study was registered at www.clinicaltrials.gov as #NCT02400437.
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- 2020
18. Genomic Landscape of Waldenström Macroglobulinemia and Its Impact on Treatment Strategies
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Amanda Kofides, Nicholas Tsakmaklis, Irene M. Ghobrial, Zachary R. Hunter, Jorge J. Castillo, Romanos Sklavenitis-Pistofidis, Andrew Keezer, Xia Liu, Antonio Sacco, Gloria Chan, Jiaji G. Chen, Steven P. Treon, Christopher J. Patterson, Cristina Jimenez, Guang Yang, Lian Xu, Mark Bustoros, Maria Luisa Guerrera, Aldo M. Roccaro, Maria Demos, Joshua Gustine, Kirsten Meid, Andrew R. Branagan, and Manit Munshi
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Cancer Research ,Receptors, CXCR4 ,ARID1A ,DNA Copy Number Variations ,medicine.disease_cause ,CXCR4 ,chemistry.chemical_compound ,immune system diseases ,hemic and lymphatic diseases ,Medicine ,Bruton's tyrosine kinase ,Humans ,Pathology, Molecular ,Review Articles ,Mutation ,biology ,business.industry ,Waldenstrom macroglobulinemia ,Genomics ,CD79B ,medicine.disease ,Lymphoma ,Oncology ,chemistry ,Ibrutinib ,Myeloid Differentiation Factor 88 ,biology.protein ,Cancer research ,Waldenstrom Macroglobulinemia ,business - Abstract
Next-generation sequencing has revealed recurring somatic mutations in Waldenström macroglobulinemia (WM), including MYD88 (95%-97%), CXCR4 (30%-40%), ARID1A (17%), and CD79B (8%-15%). Deletions involving chromosome 6q are common in patients with mutated MYD88 and include genes that modulate NFKB, BCL2, Bruton tyrosine kinase (BTK), and apoptosis. Patients with wild-type MYD88 WM show an increased risk of transformation and death and exhibit many mutations found in diffuse large B-cell lymphoma. The discovery of MYD88 and CXCR4 mutations in WM has facilitated rational drug development, including the development of BTK and CXCR4 inhibitors. Responses to many agents commonly used to treat WM, including the BTK inhibitor ibrutinib, are affected by MYD88 and/or CXCR4 mutation status. The mutation status of both MYD88 and CXCR4 can be used for a precision-guided treatment approach to WM.
- Published
- 2020
19. <scp>CXCR4</scp> mutational status does not impact outcomes in patients with <scp>W</scp> aldenström macroglobulinemia treated with proteasome inhibitors
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Christopher J. Patterson, Lian Xu, Nicholas Tsakmaklis, Guang Yang, Maria Luisa Guerrera, Maria Demos, Kirsten Meid, Amanda Kofides, Zachary R. Hunter, Jorge J. Castillo, Xia Liu, Manit Munshi, Steven P. Treon, Catherine A Flynn, Cristina Jimenez, and Joshua Gustine
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Male ,Oncology ,Receptors, CXCR4 ,medicine.medical_specialty ,MEDLINE ,Kaplan-Meier Estimate ,CXCR4 ,Text mining ,Internal medicine ,medicine ,Humans ,Mutational status ,In patient ,Prospective Studies ,Aged ,Proportional Hazards Models ,Aged, 80 and over ,Clinical Trials as Topic ,business.industry ,Waldenstrom macroglobulinemia ,Hematology ,Middle Aged ,Prognosis ,medicine.disease ,Progression-Free Survival ,Logistic Models ,Treatment Outcome ,Proteasome ,Mutation ,Myeloid Differentiation Factor 88 ,Female ,Waldenstrom Macroglobulinemia ,beta 2-Microglobulin ,business ,Proteasome Inhibitors ,Follow-Up Studies - Published
- 2020
20. Ibrutinib Monotherapy in Symptomatic, Treatment-Naïve Patients With Waldenström Macroglobulinemia
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Nicholas Tsakmaklis, Toni Dubeau, Manit Munshi, Zachary R. Hunter, Maria Demos, Guang Yang, Andrew Yee, Steven P. Treon, Xia Liu, Lian Xu, Amanda Kofides, Joshua Gustine, Jorge J. Castillo, Elizabeth O'Donnell, Gloria Chan, Noopur Raje, Kirsten Meid, and Jiaji G. Chen
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Adult ,Male ,Receptors, CXCR4 ,Cancer Research ,medicine.medical_specialty ,Urinary system ,Antineoplastic Agents ,Neutropenia ,Gastroenterology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Piperidines ,Internal medicine ,medicine ,Humans ,Prospective cohort study ,Aged ,Aged, 80 and over ,business.industry ,Adenine ,Waldenstrom macroglobulinemia ,Atrial fibrillation ,Middle Aged ,medicine.disease ,Pyrimidines ,Upper respiratory tract infection ,Oncology ,chemistry ,030220 oncology & carcinogenesis ,Ibrutinib ,Mutation ,Myeloid Differentiation Factor 88 ,Toxicity ,Pyrazoles ,Female ,Waldenstrom Macroglobulinemia ,business ,030215 immunology - Abstract
Purpose Ibrutinib is active in previously treated Waldenström macroglobulinemia (WM). MYD88 mutations ( MYD88MUT) and CXCR4 mutations ( CXCR4MUT) affect ibrutinib response. We report on a prospective study of ibrutinib monotherapy in symptomatic, untreated patients with WM, and the effect of CXCR4MUT status on outcome. Patients and Methods Symptomatic, treatment-naïve patients with WM were eligible. Ibrutinib (420 mg) was administered daily until progression or unacceptable toxicity. All tumors were genotyped for MYD88MUT and CXCR4MUT. Results A total of 30 patients with WM received ibrutinib. All carried MYD88MUT, and 14 (47%) carried a CXCR4MUT. After ibrutinib treatment, median serum IgM levels declined from 4,370 to 1,513 mg/dL, bone marrow involvement declined from 65% to 20%, and hemoglobin level rose from 10.3 to 13.9 g/dL ( P < .001 for all comparisons). Overall (minor or more than minor) and major (partial or greater than partial) responses for all patients were 100% and 83%, respectively. Rates of major (94% v 71%) and very good partial (31 v 7%) responses were higher and time to major responses more rapid (1.8 v 7.3 months; P = 0.01) in patients with wild-type CXCR4 versus those with CXCR4MUT, respectively. With a median follow-up of 14.6 months, disease in two patients (both with CXCR4MUT) progressed. The 18-month, estimated progression-free survival is 92% (95% CI, 73% to 98%). All patients are alive. Grade 2/3 treatment-related toxicities in > 5% of patients included arthralgia (7%), bruising (7%), neutropenia (7%), upper respiratory tract infection (7%), urinary tract infection (7%), atrial fibrillation (10%), and hypertension (13%). There were no grade 4 or unexpected toxicities. Conclusion Ibrutinib is highly active, produces durable responses, and is safe as primary therapy in patients with symptomatic WM. CXCR4MUT status affects responses to ibrutinib.
- Published
- 2018
21. BTKCys481Ser drives ibrutinib resistance via ERK1/2 and protects BTKwild-type MYD88-mutated cells by a paracrine mechanism
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Sara J. Buhrlage, Toni Dubeau, Zachary R. Hunter, Patricia Severns, Amanda Kofides, Maria Demos, Maria Luisa Guerrera, Nathanael S. Gray, Guang Yang, Christopher J. Patterson, Steven P. Treon, Kirsten Meid, Jinhua Wang, Nicholas Tsakmaklis, Jorge J. Castillo, Gloria Chan, Xia Liu, Manit Munshi, Lian Xu, Joshua Gustine, and Jiaji G. Chen
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0301 basic medicine ,Chemistry ,medicine.medical_treatment ,Immunology ,Waldenstrom macroglobulinemia ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Proinflammatory cytokine ,03 medical and health sciences ,Interleukin 10 ,chemistry.chemical_compound ,Paracrine signalling ,030104 developmental biology ,0302 clinical medicine ,Cytokine ,Cell culture ,030220 oncology & carcinogenesis ,Ibrutinib ,Cancer research ,medicine ,Diffuse large B-cell lymphoma - Abstract
Acquired ibrutinib resistance due to BTKCys481 mutations occurs in B-cell malignancies, including those with MYD88 mutations. BTKCys481 mutations are usually subclonal, and their relevance to clinical progression remains unclear. Moreover, the signaling pathways that promote ibrutinib resistance remain to be clarified. We therefore engineered BTKCys481Ser and BTKWT expressing MYD88-mutated Waldenstrom macroglobulinemia (WM) and activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL) cells and observed reactivation of BTK-PLCγ2-ERK1/2 signaling in the presence of ibrutinib in only the former. Use of ERK1/2 inhibitors triggered apoptosis in BTKCys481Ser-expressing cells and showed synergistic cytotoxicity with ibrutinib. ERK1/2 reactivation in ibrutinib-treated BTKCys481Ser cells was accompanied by release of many prosurvival and inflammatory cytokines, including interleukin-6 (IL-6) and IL-10 that were also blocked by ERK1/2 inhibition. To clarify if cytokine release by ibrutinib-treated BTKCys481Ser cells could protect BTKWT MYD88-mutated malignant cells, we used a Transwell coculture system and showed that nontransduced BTKWT MYD88-mutated WM or ABC DLBCL cells were rescued from ibrutinib-induced killing when cocultured with BTKCys481Ser but not their BTKWT-expressing counterparts. Use of IL-6 and/or IL-10 blocking antibodies abolished the protective effect conferred on nontransduced BTKWT by coculture with BTKCys481Ser expressing WM or ABC DLBCL cell counterparts. Rebound of IL-6 and IL-10 serum levels also accompanied disease progression in WM patients with acquired BTKCys481 mutations. Our findings show that the BTKCys481Ser mutation drives ibrutinib resistance in MYD88-mutated WM and ABC DLBCL cells through reactivation of ERK1/2 and can confer a protective effect on BTKWT cells through a paracrine mechanism.
- Published
- 2018
22. Deepening of response after completing rituximab-containing therapy in patients with Waldenstrom macroglobulinemia
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Maria Demos, Catherine A Flynn, Xia Liu, Lian Xu, Christopher J. Patterson, Amanda Kofides, Jiaji Chen, Gloria Chan, Jorge J. Castillo, Manit Munshi, Kirsten Meid, Maria Luisa Guerrera, Cristina Jimenez, Joshua Gustine, Steven P. Treon, Toni Dubeau, Zachary R. Hunter, Andrew Keezer, Nicholas Tsakmaklis, and Guang Yang
- Subjects
Adult ,Male ,medicine.medical_specialty ,Receptors, CXCR4 ,CXCR4 ,Maintenance Chemotherapy ,03 medical and health sciences ,0302 clinical medicine ,Maintenance therapy ,Bone Marrow ,Internal medicine ,Antineoplastic Combined Chemotherapy Protocols ,Medicine ,Humans ,Progression-free survival ,Survival analysis ,Aged ,Retrospective Studies ,Aged, 80 and over ,business.industry ,Waldenstrom macroglobulinemia ,Retrospective cohort study ,Hematology ,Middle Aged ,medicine.disease ,Survival Analysis ,Progression-Free Survival ,medicine.anatomical_structure ,Immunoglobulin M ,030220 oncology & carcinogenesis ,Myeloid Differentiation Factor 88 ,Rituximab ,Female ,Bone marrow ,Waldenstrom Macroglobulinemia ,business ,030215 immunology ,medicine.drug ,Follow-Up Studies ,Paraproteins - Abstract
Rituximab-containing regimens are commonly used for frontline therapy in patients with symptomatic Waldenstrom macroglobulinemia (WM). We had observed that a portion of WM patients experienced deepening of response months to years after therapy completion. We carried a retrospective study aimed at describing this phenomenon. We gathered baseline data, and responses at end of induction, end of maintenance and best response. Deepening of response was defined as ≥25% decrease in serum IgM achieved at a later time from therapy completion. Of 178 patients included, 116 (65%) received maintenance therapy and 62 (35%) were observed. In patients who received maintenance, 44 (38%) had ≥25% decrease in serum IgM level after the end of maintenance with a median time from end of maintenance to lowest IgM level of 1.6 years (range 0.1-7.9 years). In patients who were observed, 19 (31%) had ≥25% decrease in serum IgM level after the end of induction with a median time from end of induction to lowest IgM level of 1.6 years (range 0.2-5.1 years). Baseline hemoglobin
- Published
- 2019
23. CXCR4 mutation subtypes impact response and survival outcomes in patients with Waldenström macroglobulinaemia treated with ibrutinib
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Jiaji G. Chen, Steven P. Treon, Manit Munshi, Gloria Chan, Nicholas Tsakmaklis, Andrew Keezer, Maria Demos, Christopher J. Patterson, Amanda Kofides, Xia Liu, Maria Luisa Guerrera, Kirsten Meid, Guang Yang, Jorge J. Castillo, Lian Xu, Toni Dubeau, Zachary R. Hunter, and Joshua Gustine
- Subjects
Oncology ,Adult ,Male ,medicine.medical_specialty ,Receptors, CXCR4 ,medicine.disease_cause ,CXCR4 ,Disease-Free Survival ,Frameshift mutation ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Piperidines ,Internal medicine ,medicine ,Humans ,Aged ,Response rate (survey) ,Aged, 80 and over ,Mutation ,business.industry ,Adenine ,Hazard ratio ,Hematology ,Odds ratio ,Middle Aged ,Confidence interval ,Neoplasm Proteins ,Survival Rate ,Pyrimidines ,chemistry ,030220 oncology & carcinogenesis ,Ibrutinib ,Pyrazoles ,Female ,Waldenstrom Macroglobulinemia ,business ,030215 immunology - Abstract
Ibrutinib is associated with response rate of 90% and median progression-free survival (PFS) in excess of 5 years in Waldenstrom macroglobulinaemia (WM) patients. CXCR4 mutations are detected in 30-40% of patients with WM and associate with lower rates of response and shorter PFS to ibrutinib therapy. Both frameshift (CXCR4FS ) and nonsense (CXCR4NS ) CXCR4 mutations have been described. The impact of these mutations on outcomes to ibrutinib have not been evaluated in WM patients. We studied consecutive patients with a diagnosis of WM, on ibrutinib therapy, for the presence of CXCR4FS and CXCR4NS mutations and evaluated the differences in response and PFS between groups. Of 180 patients, 68 patients (38%) had CXCR4 mutations; 49 (27%) had CXCR4NS and 19 (11%) had CXCR4FS mutations. In multivariate models, patients with CXCR4NS had lower odds of major response (Odds ratio 0·25, 95% confidence interval [CI] 0·12-0·53; P
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- 2019
24. Abstract IA39: Investigating malignant transformation in Waldenstrom's macroglobulinemia
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Steven P. Treon, Zachary R. Hunter, Nicholas Tsakmaklis, Maria Luisa Guerrera, Xia Lu, and Guang Yang
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Mutation ,education.field_of_study ,Somatic cell ,Population ,Macroglobulinemia ,General Medicine ,Biology ,medicine.disease_cause ,medicine.disease ,CD19 ,Frameshift mutation ,Malignant transformation ,Lymphoma ,medicine ,Cancer research ,biology.protein ,education - Abstract
Waldenström's macroglobulinemia (WM) is an uncommon B-cell lymphoma that corresponds to the histopathologic classification of IgM-secreting lymphomplasmacytic lymphoma. It is also a disease characterized by a series of highly recurrent mutations. Whole-genome sequencing of CD19+ bone marrow cells from patients with WM led to the discovery of a somatic heterozygous c.978T>C mutation in Toll-like receptor adaptor protein MYD88, resulting in a leucine-to-proline substitution p.Leu265Pro (L265P) in over 90% of WM patients. Somatic activating mutations in MYD88 induce constitutive homodimerization and downstream signaling through the nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB) pathway independent of receptor activation. Other somatic events characteristic of WM include deletions in chromosome 6q21-23 and activating nonsense and/or frameshift mutations in the carboxyl-terminal tail of CXCR4, found in 40-60% and over 30% of WM patients, respectively. Both of these events are found predominantly in MYD88 mutant WM and are frequently restricted to a subclonal population. Similar to related lymphomas, WM is thought to evolve from IgM-secreting monoclonal gammopathy of undermined significance (MGUS). The MYD88 mutations are easily detectible IgM MGUS stage, suggesting that it is a very early event in clonal development but may predate the malignant transformation into WM. This theory is support by several transgenic mouse models of mutant MYD88 that have demonstrated that p.Leu265Pro MYD88 mutation in B cells alone is not sufficient for lymphomagenesis (Knittel et al., Blood 2016; Sewastianik et al., Blood Adv 2019). Mutations in CXCR4 have been detected in IgM MGUS, though at lower rates than are typical of WM. Given that this and the transcriptional profile of CXCR4 activating mutations in WM are consistent with a pattern of suppression of tumor suppressors upregulated by mutant MYD88, it has been proposed as a possible transformation event from IgM MGUS to asymptomatic WM. Likewise, a number of careful genetic studies of IgM MGUS have suggested the emergence of somatic copy number alterations, particularly the deletions in 6q, to be related to the malignant transformation of WM (Schop et al., Cancer Genet Cytogenet 2006; Paiva et al., Blood 2015). We therefore reanalyzed our whole-genome sequencing data of WM patients and performed allele-specific PCR and 6q copy number assays to study the rates and relationship of CXCR4 mutations and chromosome 6q deletions in untreated MYD88 mutated WM. Both our 30-patient whole-genome and our 25-patient PCR cohort found no relationship between symptomatic and asymptomatic WM and the somatic events, indicating that they both were occurring at an earlier stage of WM development. Both studies also found that the large clonal deletions in chromosome 6q were not observed in the presence of CXCR4 mutations (p Citation Format: Zachary R. Hunter, Maria Luisa Guerrera, Guang Yang, Nicholas Tsakmaklis, Xia Lu, Steven P. Treon. Investigating malignant transformation in Waldenstrom's macroglobulinemia [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA39.
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- 2020
25. Transcriptome sequencing reveals a profile that corresponds to genomic variants in Waldenström macroglobulinemia
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Jie Chen, Nikhil M. Munshi, Christopher J. Patterson, Josephine M.I. Vos, Jorge J. Castillo, Kenneth C. Anderson, Guang Yang, Joshua Gustine, Lian Xu, Robert Manning, Philip Brodsky, Nicholas Tsakmaklis, Toni Dubeau, Zachary R. Hunter, Jiaji G. Chen, Steven P. Treon, and Xia Liu
- Subjects
0301 basic medicine ,Genetics ,Lymphoid Neoplasia ,biology ,Immunology ,Waldenstrom macroglobulinemia ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,CXCR4 ,Recombination-activating gene ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,RAG2 ,030220 oncology & carcinogenesis ,Gene expression ,medicine ,Activation-induced (cytidine) deaminase ,biology.protein ,Transcriptional regulation ,Gene - Abstract
Whole-genome sequencing has identified highly prevalent somatic mutations including MYD88, CXCR4, and ARID1A in Waldenstrom macroglobulinemia (WM). The impact of these and other somatic mutations on transcriptional regulation in WM remains to be clarified. We performed next-generation transcriptional profiling in 57 WM patients and compared findings to healthy donor B cells. Compared with healthy donors, WM patient samples showed greatly enhanced expression of the VDJ recombination genes DNTT, RAG1, and RAG2, but not AICDA. Genes related to CXCR4 signaling were also upregulated and included CXCR4, CXCL12, and VCAM1 regardless of CXCR4 mutation status, indicating a potential role for CXCR4 signaling in all WM patients. The WM transcriptional profile was equally dissimilar to healthy memory B cells and circulating B cells likely due increased differentiation rather than cellular origin. The profile for CXCR4 mutations corresponded to diminished B-cell differentiation and suppression of tumor suppressors upregulated by MYD88 mutations in a manner associated with the suppression of TLR4 signaling relative to those mutated for MYD88 alone. Promoter methylation studies of top findings failed to explain this suppressive effect but identified aberrant methylation patterns in MYD88 wild-type patients. CXCR4 and MYD88 transcription were negatively correlated, demonstrated allele-specific transcription bias, and, along with CXCL13, were associated with bone marrow disease involvement. Distinct gene expression profiles for patients with wild-type MYD88, mutated ARID1A, familial predisposition to WM, chr6q deletions, chr3q amplifications, and trisomy 4 are also described. The findings provide novel insights into the molecular pathogenesis and opportunities for targeted therapeutic strategies for WM.
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- 2016
26. Targeting Myddosome Assembly in Waldenstrom Macroglobulinaemia
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Christopher J. Patterson, Sara J. Buhrlage, Steven P. Treon, Nathanael S. Gray, Guang Yang, Nicholas Tsakmaklis, Jie Chen, Xia Liu, Jiaji G. Chen, Jorge J. Castillo, Lian Xu, and Zachary R. Hunter
- Subjects
Models, Molecular ,0301 basic medicine ,Molecular Conformation ,Antineoplastic Agents ,Structure-Activity Relationship ,03 medical and health sciences ,0302 clinical medicine ,Drug Discovery ,Humans ,Bruton's tyrosine kinase ,Protein Interaction Domains and Motifs ,Molecular Targeted Therapy ,Waldenström macroglobulinaemia ,biology ,Hematology ,Peptide Fragments ,030104 developmental biology ,030220 oncology & carcinogenesis ,Mutation ,Myeloid Differentiation Factor 88 ,Cancer research ,biology.protein ,Protein Multimerization ,Waldenstrom Macroglobulinemia ,Protein Binding ,Signal Transduction - Published
- 2016
27. Spotting the elusive Siberian tiger: Complete response to ibrutinib in a patient with Waldenström macroglobulinemia
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Steven P. Treon, Toni Dubeau, Zachary R. Hunter, Nicholas Tsakmaklis, Amanda Kofides, Maria Demos, Lian Xu, and Jorge J. Castillo
- Subjects
Oncology ,medicine.medical_specialty ,biology ,business.industry ,Waldenstrom macroglobulinemia ,Hematology ,Spotting ,medicine.disease ,biology.organism_classification ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,chemistry ,030220 oncology & carcinogenesis ,Internal medicine ,Ibrutinib ,medicine ,business ,Complete response ,Siberian tiger ,030215 immunology - Published
- 2018
28. A Novel HCK and BTK Dual Inhibitor Kin-8194 Shows Superior Activity over Ibrutinib and Overcomes BTKC481S Mediated Ibrutinib Resistance in Vitro and In Vivo in MYD88 Mutated B-Cell Lymphomas
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Gloria Chan, Jinhua Wang, Amanda Kofides, Jorge J. Castillo, Nathanael S. Gray, Jiaji G. Chen, Nicholas Tsakmaklis, Christopher J. Patterson, Maria Demos, Guang Yang, Sara J. Buhrlage, Maria Luisa Guerrera, Zachary R. Hunter, Xia Liu, Steven P. Treon, Cristina Jimenez, Lian Xu, and Manit Munshi
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Mutation ,biology ,Chemistry ,Immunology ,Dual inhibitor ,Cell Biology ,Hematology ,medicine.disease ,medicine.disease_cause ,Biochemistry ,In vitro ,chemistry.chemical_compound ,medicine.anatomical_structure ,In vivo ,Ibrutinib ,medicine ,Cancer research ,biology.protein ,Bruton's tyrosine kinase ,Diffuse large B-cell lymphoma ,B cell - Abstract
Activating mutations in MYD88 promote malignant cell growth and survival through multiple pathways that include BTK and HCK. HCK is transcriptionally upregulated and activated by mutated MYD88 and in turn activates BTK itself, as well as ERK and AKT. Ibrutinib is a covalent inhibitor that binds to BTKCys481 and shows activity in MYD88 mutated B-cell malignancies, including WM, MZL, ABC DLBCL, and PCNSL. Resistance to ibrutinib on the basis of BTKCys481 as well as downstream mutations is increasingly being recognized. We therefore sought to develop potent and selective inhibitors that target HCK. We developed a non-covalent dual inhibitor, KIN-8194, of HCK and BTK following a screen >220 clinical and preclinical kinase inhibitors, and lead optimization following synthesis >400 analogs. Dual HCK and BTK inhibition was confirmed by both KINOMEscan® and live-cell target engagement studies (KiNativ™ profiling and live cell ATP-biotin competition assay). KIN-8194 showed robust suppression of HCK (IC50 Disclosures Hunter: Janssen: Consultancy. Castillo:Beigene: Consultancy, Research Funding; TG Therapeutics: Research Funding; Abbvie: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding. Gray:Gatekeeper, Syros, Petra, C4, B2S and Soltego.: Equity Ownership; Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi.: Equity Ownership, Research Funding. Treon:Pharmacyclics: Research Funding; BMS: Research Funding; Janssen: Consultancy.
- Published
- 2019
29. CXCR4 Mutational Status Does Not Impact Outcomes in Patients with Waldenstrom Macroglobulinemia Treated with Proteasome Inhibitors
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Gloria Chan, Steven P. Treon, Maria Demos, Manit Munshi, Kirsten Meid, Xia Liu, Andrew Keezer, Lian Xu, Amanda Kofides, Jiaji Chen, Nicholas Tsakmaklis, Joshua Gustine, Toni Dubeau, Zachary R. Hunter, Maria Luisa Guerrera, Guang Yang, and Jorge J. Castillo
- Subjects
Oncology ,medicine.medical_specialty ,Bortezomib ,business.industry ,Immunology ,Waldenstrom macroglobulinemia ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,CXCR4 ,Carfilzomib ,Ixazomib ,chemistry.chemical_compound ,Proteasome ,chemistry ,Internal medicine ,Ibrutinib ,medicine ,Rituximab ,business ,medicine.drug - Abstract
Introduction: Approximately 40% of patients with Waldenström macroglobulinemia (WM) have an activating somatic mutation in CXCR4, including both nonsense and frameshift variants. There are limited data on the impact of CXCR4 mutations on the outcomes to therapy in WM patients. Mounting evidence suggests that CXCR4 mutations adversely affect depth of response and progression-free survival (PFS) to ibrutinib in patients with WM. Methods: We performed a pooled analysis evaluating the impact of CXCR4 mutations and CXCR4 mutation subtypes on response, PFS and survival after frontline treatment initiation (SAFTI) on 76 WM patients who received proteasome inhibitor-based primary therapy. All patients were participants on three prospective clinical trials evaluating the combinations of bortezomib, dexamethasone and rituximab (BDR; ClinicalTrials.Gov ID NCT00250926), carfilzomib, dexamethasone and rituximab (CaRD; ClinicalTrials.Gov ID NCT01470196), and ixazomib, dexamethasone and rituximab (IDR; ClinicalTrials.Gov ID NCT02400437) in previously untreated patients with WM, and were treated at the Bing Center for WM. All patients met criteria for a clinicopathological diagnosis of WM and for treatment initiation, according to the guidelines established by the 2nd International Workshop for WM (IWWM). CXCR4 mutations were divided in nonsense and frameshift mutations, as previously described. Response to therapy was assessed using response criteria by the 3rdIWWM. We fitted univariate and multivariate logistic regression and proportional-hazard Cox regression models for major response and PFS and SAFTI, respectively. P-values Results: A total of 76 patients were included in this analysis, of which 19 (25%) received BDR, 31 (41%) received CaRD and 26 (34%) received IDR. Thirty-six patients (55%) did not have CXCR4 mutations and 29 (45%) had a CXCR4 mutation, of which 16 had nonsenseand 13 had frameshift CXCR4 mutations. In 11 patients, the CXCR4 mutational status was not determined. The median age at treatment initiation was 63 years (range 46-83 years), median hemoglobin level was 10.3 g/dl (range 6.9-17.1 g/dl), median platelet count was 245 K/uL (range 68-584 K/uL), median serum IgM level was 4,048 mg/dl (range 345-9,550 mg/dl), median serum β2-microglobulin level was 3.5 mg/l (range 1.0-10.8 mg/l), serum albumin was 3.7 g/dl (range 2.4-4.8 g/dl) and median bone marrow involvement was 55% (range 5-95%). There was a higher proportion of serum IgM levels ≥4,000 mg/dl (62% vs. 39%) and lower proportion of serum albumin levels ≤3.5 g/dl (21% vs. 47%) in patients with than in patients without CXCR4 mutations. Our study showed no detectable differences in major response rate at 6 months (OR 0.77, 95% CI 0.27-2.22; p=0.63) and 12 months (OR 0.76, 95% CI 0.18-3.23; p=0.71) between patients with and without CXCR4 mutations. The median PFS for patients without CXCR4 mutations was 3.6 years (95% CI 1.7-5.9 years) versus 6.5 years (95% CI 2.7-not reached) for patients with CXCR4 mutations, which was not statistically significantly different (HR 0.59, 95% CI 0.29-1.17; p=0.13). Frameshift CXCR4 mutations were associated with better PFS than nonsense CXCR4 mutations in a multivariate Cox regression model (HR 0.22, 95% CI 0.06-0.80; p=0.02). Patients with CXCR4 mutations had a 10-year SAFTI rate of 75% (95% CI 13-96%) versus 83% (95% CI 27-97%) in patients without CXCR4 mutations, which was not statistically significantly different (HR 3.01, 95% CI 0.17-52.6; p=0.45). Conclusions: We conclude that there is no evidence that CXCR4 mutations adversely impact response, PFS and SAFTI in WM patients receiving proteasome inhibitor-based primary therapy. Figure Disclosures Castillo: Janssen: Consultancy, Research Funding; Abbvie: Research Funding; TG Therapeutics: Research Funding; Pharmacyclics: Consultancy, Research Funding; Beigene: Consultancy, Research Funding. Hunter:Janssen: Consultancy. Treon:BMS: Research Funding; Janssen: Consultancy; Pharmacyclics: Research Funding.
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- 2019
30. Clinical and Genomic Factors Are Predictive of Response and Prognostic of Progression-Free Survival in Patients with Waldenström Macroglobulinemia Treated with Ibrutinib
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Nicholas Tsakmaklis, Andrew Keezer, Jiaji G. Chen, Amanda Kofides, Xia Liu, Maria Luisa Guerrera, Steven P. Treon, Jorge J. Castillo, Gloria Chan, Maria Demos, Manit Munshi, Lian Xu, Joshua Gustine, Guang Yang, Kirsten Meid, and Zachary R. Hunter
- Subjects
Oncology ,medicine.medical_specialty ,business.industry ,Immunology ,Cancer ,Waldenstrom macroglobulinemia ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,chemistry.chemical_compound ,chemistry ,Ibrutinib ,Internal medicine ,Partial response ,medicine ,Platelet Count measurement ,In patient ,Progression-free survival ,business ,Bing–Neel syndrome - Abstract
Introduction: The Bruton tyrosine kinase inhibitor ibrutinib is the only FDA approved therapy for the treatment of symptomatic Waldenstrom macroglobulinemia (WM), and has been associated with high response rates and durable progression-free survival (PFS). Factors associated with depth of response and PFS duration are not well established. We performed a retrospective study aimed at identifying predictive and prognostic factors in WM patients treated with ibrutinib. Methods: We included consecutive patients with a diagnosis of WM treated with ibrutinib monotherapy evaluated at the Dana-Farber Cancer Institute since January 2012 through March 2019. Patients with Bing-Neel syndrome (WM involving the central nervous system) were excluded. Baseline clinical and laboratory characteristics were gathered. MYD88 and CXCR4 mutations were assessed using polymerase chain reaction assays and Sanger sequencing. Responses at 6 months were assessed using criteria from IWWM3. PFS was defined as the time from ibrutinib initiation until last follow-up, death or progression. Univariate and multivariate logistic regression models were fitted for partial response (PR) and very good partial response (VGPR) at 6 months, and Cox proportional-hazard regression models were fitted for PFS. Results: A total of 252 patients were included in our analysis. Selected baseline characteristics include: age ≥65 years (60%), hemoglobin Conclusion: Serum albumin and CXCR4 mutations emerge as important factors predictive of PR at 6 months and also prognostic of PFS in WM patients treated with ibrutinib. A novel PFS stratification tool that separates patients into 3 risk groups was established and would need further validation. Figure Disclosures Castillo: Abbvie: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Beigene: Consultancy, Research Funding; TG Therapeutics: Research Funding. Hunter:Janssen: Consultancy. Treon:Pharmacyclics: Research Funding; BMS: Research Funding; Janssen: Consultancy.
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- 2019
31. Mutated MYD88 Regulates HCK Pro-Survival Signaling through JunB in MYD88 Mutated B-Lymphoma Cells
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Jorge J. Castillo, Andrew Keezer, Gloria Chan, Lian Xu, Nicholas Tsakmaklis, Kirsten Meid, Maria Luisa Guerrera, Xia Liu, Zachary R. Hunter, Amanda Kofides, Christopher J. Patterson, Steven P. Treon, Cristina Jimenez, Manit Munshi, Jiaji G. Chen, Maria Demos, and Guang Yang
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biology ,JUNB ,Immunology ,hemic and immune systems ,Cell Biology ,Hematology ,Biochemistry ,hemic and lymphatic diseases ,biology.protein ,Cancer research ,Transcriptional regulation ,Phosphorylation ,Bruton's tyrosine kinase ,Signal transduction ,STAT3 ,Protein kinase B ,Transcription factor - Abstract
Hematopoietic cell kinase (HCK) is a member of the SRC family tyrosine kinases (SFKs) that is down-regulated in later stages of B-cell ontogeny. In MYD88 mutated B-cell lymphomas, HCK is aberrantly over-expressed and is activated and triggers multiple growth and survival pathways including BTK, PI3Kδ/AKT and ERK1/2 which are essential to tumor cell survival. Ibrutinib, a pleiotropic inhibitor of BTK, that produces remarkable responses in MYD88 mutated WM, ABC-DLBCL, and Primary CNS Lymphoma was found also potently inhibits HCK. Mutations that abolish ibrutinib-HCK binding greatly diminish anti-tumor activity in MYD88 mutated lymphoma cells, highlighting the importance of HCK as an essential target in MYD88 driven diseases. To clarify the transcriptional regulation of HCK in MYD88 mutated malignancies, we performed promoter binding TF profiling, PROMO weighted transcription factor (TF) consensus binding analysis, and chromatin immuno-precipitation (ChIP) studies. We identified PAX5, and mutated MYD88 downstream signaling mediators, STAT3, AP-1 and NF-kB as important drivers of HCK transcription. Knockdown of PAX5, a crucial regulatory factor required for B-cell commitment and identity, abrogated HCK transcription in MYD88 mutated lymphoma cells. Deletion of STAT3, AP-1 or NF-kB binding sites greatly reduced corresponding TFs binding and HCK promoter activity, indicating the importance of MYD88 directed signaling in the regulation of HCK transcription. Among AP-1 complex components, JunB but not c-Jun showed greatest relevance to TLR/MYD88 signaling and regulation of HCK transcription. Since STAT3 and NF-kB are known downstream mediators of mutated MYD88 signaling, we focused on the function of JunB. JunB phosphorylation increased following MYD88 pathway activation through TLR4 (with LPS-EB) or TLR9 (with ODN-2006), as well as lentiviral mediated expression of mutated MYD88 (L265P) but not wild type MYD88 (MYD88-WT) at Thr102 and Thr104 in either MYD88 mutated BCWM.1 cells or MYD88-WT Ramos cells. Conversely, c-Jun phosphorylation was not impacted by either intervention. Knockdown of MYD88 in BCWM.1 WM cells also reduced the phosphorylation of JunB, while c-Jun phosphorylation showed modest increase. Moreover, knockdown of JunB reduced HCK protein levels in MYD88 mutated WM and ABC-DLBCL cells. These data demonstrate that JunB is an important mediator of mutated MYD88 signaling among AP1 complex members and is directly involved in the regulation of HCK expression in MYD88 mutated B-cell lymphoma. The findings provide new insights into the transcriptional regulation of HCK by MYD88 driven TFs, and opportunities for further advancing targeted therapeutics in MYD88 driven B-cell malignancies. Disclosures Hunter: Janssen: Consultancy. Castillo:TG Therapeutics: Research Funding; Pharmacyclics: Consultancy, Research Funding; Beigene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Abbvie: Research Funding. Treon:Pharmacyclics: Research Funding; BMS: Research Funding; Janssen: Consultancy.
- Published
- 2019
32. TP53 mutations are associated with mutated MYD88 and CXCR4, and confer an adverse outcome in Waldenström macroglobulinaemia
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Jiaji G. Chen, Lian Xu, Maria Demos, Toni Dubeau, Zachary R. Hunter, C. J. Patterson, Manit Munshi, Kirsten Meid, Gloria Chan, Xia Liu, Maria Luisa Guerrera, Amanda Kofides, Steven P. Treon, Guang Yang, Joshua Gustine, Jorge J. Castillo, and Nicholas Tsakmaklis
- Subjects
Oncology ,Adult ,Male ,medicine.medical_specialty ,Receptors, CXCR4 ,endocrine system diseases ,Adverse outcomes ,Tp53 mutation ,CXCR4 ,Disease-Free Survival ,03 medical and health sciences ,symbols.namesake ,chemistry.chemical_compound ,0302 clinical medicine ,stomatognathic system ,Piperidines ,Internal medicine ,medicine ,Humans ,In patient ,Waldenström macroglobulinaemia ,neoplasms ,Aged ,Sanger sequencing ,business.industry ,Adenine ,Hematology ,Middle Aged ,medicine.disease ,Survival Rate ,Pyrimidines ,chemistry ,030220 oncology & carcinogenesis ,Ibrutinib ,Mutation ,Myeloid Differentiation Factor 88 ,symbols ,Pyrazoles ,Female ,Tumor Suppressor Protein p53 ,Waldenstrom Macroglobulinemia ,business ,Progressive disease ,030215 immunology ,Follow-Up Studies - Abstract
Little is known about TP53 mutations in Waldenstrom Macroglobulinaemia (WM). We evaluated 265 WM patients for TP53 mutations by next-generation sequencing, and validated the findings by Sanger sequencing. TP53 mutations were identified and validated in 6 (2·6%) patients that impacted the DNA-binding domain. All six were MYD88- and CXCR4-mutated. Ibrutinib showed activity in patients carrying all three mutations. With a median follow-up of 18 months, 2 (33%) with biallelic TP53 inactivation died of progressive disease. TP53 mutations are rare in WM, and associate with MYD88 and CXCR4 mutations. WM patients with TP53 mutations show response to ibrutinib.
- Published
- 2018
33. BTK
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Jiaji G, Chen, Xia, Liu, Manit, Munshi, Lian, Xu, Nicholas, Tsakmaklis, Maria G, Demos, Amanda, Kofides, Maria Luisa, Guerrera, Gloria G, Chan, Christopher J, Patterson, Kirsten, Meid, Joshua, Gustine, Toni, Dubeau, Patricia, Severns, Jorge J, Castillo, Zachary R, Hunter, Jinhua, Wang, Sara J, Buhrlage, Nathanael S, Gray, Steven P, Treon, and Guang, Yang
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Cell Survival ,MAP Kinase Signaling System ,Adenine ,Apoptosis ,Pyrimidines ,Piperidines ,Drug Resistance, Neoplasm ,Cell Line, Tumor ,Mutation ,Myeloid Differentiation Factor 88 ,Paracrine Communication ,Agammaglobulinaemia Tyrosine Kinase ,Cytokines ,Humans ,Pyrazoles ,Inflammation Mediators - Abstract
Acquired ibrutinib resistance due to BTK
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- 2017
34. MYD88 wild-type Waldenstrom Macroglobulinaemia: differential diagnosis, risk of histological transformation, and overall survival
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Maria Demos, Maria Luisa Guerrera, Lian Xu, Christopher J. Patterson, Toni Dubeau, Guang Yang, Jiaji Chen, Manit Munshi, Zachary R. Hunter, Jorge J. Castillo, Robert Manning, Amanda Kofides, Steven P. Treon, Joshua Gustine, Kirsten Meid, Xia Liu, Patricia Severns, Nicholas Tsakmaklis, and Gloria Chan
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Adult ,Male ,medicine.medical_specialty ,Multivariate analysis ,Genotype ,DNA Mutational Analysis ,Kaplan-Meier Estimate ,Gastroenterology ,Immunophenotyping ,Diagnosis, Differential ,03 medical and health sciences ,0302 clinical medicine ,Bone Marrow ,Internal medicine ,medicine ,Humans ,Pathological ,Multiple myeloma ,Aged ,Proportional Hazards Models ,Aged, 80 and over ,business.industry ,Incidence (epidemiology) ,Hematology ,Odds ratio ,Middle Aged ,medicine.disease ,Prognosis ,Confidence interval ,Lymphoma ,Cell Transformation, Neoplastic ,030220 oncology & carcinogenesis ,Mutation ,Myeloid Differentiation Factor 88 ,Female ,Differential diagnosis ,Waldenstrom Macroglobulinemia ,business ,Biomarkers ,030215 immunology - Abstract
MYD88 mutations are present in 95% of Waldenstrom Macroglobulinaemia (WM) patients, and support diagnostic discrimination from other IgM-secreting B-cell malignancies. Diagnostic discrimination can be difficult among suspected wild-type MYD88 (MYD88WT ) WM cases. We systematically reviewed the clinical, pathological and laboratory studies for 64 suspected MYD88WT WM patients. World Health Organization and WM consensus guidelines were used to establish clinicopathological diagnosis. Up to 30% of suspected MYD88WT WM cases had an alternative clinicopathological diagnosis, including IgM multiple myeloma. The estimated 10-year survival was 73% (95% confidence interval [CI] 52-86%) for MYD88WT versus 90% (95% CI 82-95%) for mutated (MYD88MUT ) WM patients (Log-rank P < 0·001). Multivariate analysis only showed MYD88 mutation status (P < 0·001) as a significant determinant for overall survival. Diffuse large B-cell lymphoma (DLBCL) was diagnosed in 7 (15·2%) and 2 (0·76%) of MYD88WT and MYD88MUT patients, respectively (Odds ratio 23·3; 95% CI 4·2-233·8; P < 0·001). Overall survival was shorter among MYD88WT patients with an associated DLBCL event (Log-rank P = 0·08). The findings show that among suspected MYD88WT WM cases, an alternative clinicopathological diagnosis is common and can impact clinical care. WM patients with MYD88WT disease have a high incidence of associated DLBCL events and significantly shorter survival versus those with MYD88MUT disease.
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- 2017
35. Acquired mutations associated with ibrutinib resistance in Waldenström macroglobulinemia
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Christopher J. Patterson, Nicholas Tsakmaklis, Toni Dubeau, Richard R. Furman, Zachary R. Hunter, Lian Xu, M. Lia Palomba, Guang Yang, Xia Liu, Joshua Gustine, Amanda Kofides, Jorge J. Castillo, Steven P. Treon, Kirsten Meid, Jiaji G. Chen, Maria Demos, and Ranjana H. Advani
- Subjects
0301 basic medicine ,Male ,Immunology ,Biology ,medicine.disease_cause ,Biochemistry ,CXCR4 ,CD19 ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Piperidines ,immune system diseases ,hemic and lymphatic diseases ,medicine ,Bruton's tyrosine kinase ,Humans ,Tumor marker ,Aged ,Mutation ,Lymphoid Neoplasia ,Adenine ,Waldenstrom macroglobulinemia ,Cell Biology ,Hematology ,Middle Aged ,medicine.disease ,Neoplasm Proteins ,030104 developmental biology ,Pyrimidines ,chemistry ,Drug Resistance, Neoplasm ,030220 oncology & carcinogenesis ,Ibrutinib ,Cancer research ,biology.protein ,Pyrazoles ,Female ,Waldenstrom Macroglobulinemia ,Progressive disease ,Signal Transduction - Abstract
Ibrutinib produces high response rates and durable remissions in Waldenström macroglobulinemia (WM) that are impacted by MYD88 and CXCR4(WHIM) mutations. Disease progression can develop on ibrutinib, although the molecular basis remains to be clarified. We sequenced sorted CD19(+) lymphoplasmacytic cells from 6 WM patients who progressed after achieving major responses on ibrutinib using Sanger, TA cloning and sequencing, and highly sensitive and allele-specific polymerase chain reaction (AS-PCR) assays that we developed for Bruton tyrosine kinase (BTK) mutations. AS-PCR assays were used to screen patients with and without progressive disease on ibrutinib, and ibrutinib-naïve disease. Targeted next-generation sequencing was used to validate AS-PCR findings, assess for other BTK mutations, and other targets in B-cell receptor and MYD88 signaling. Among the 6 progressing patients, 3 had BTK(Cys481) variants that included BTK(Cys481Ser(c.1635G>C and c.1634T>A)) and BTK(Cys481Arg(c.1634T>C)). Two of these patients had multiple BTK mutations. Screening of 38 additional patients on ibrutinib without clinical progression identified BTK(Cys481) mutations in 2 (5.1%) individuals, both of whom subsequently progressed. BTK(Cys481) mutations were not detected in baseline samples or in 100 ibrutinib-naive WM patients. Using mutated MYD88 as a tumor marker, BTK(Cys481) mutations were subclonal, with a highly variable clonal distribution. Targeted deep-sequencing confirmed AS-PCR findings, and identified an additional BTK(Cys481Tyr(c.1634G>A)) mutation in the 2 patients with multiple other BTK(Cys481) mutations, as well as CARD11(Leu878Phe(c.2632C>T)) and PLCγ2(Tyr495His(c.1483T>C)) mutations. Four of the 5 patients with BTK(C481) variants were CXCR4 mutated. BTK(Cys481) mutations are common in WM patients with clinical progression on ibrutinib, and are associated with mutated CXCR4.
- Published
- 2017
36. HCK is a survival determinant transactivated by mutated MYD88, and a direct target of ibrutinib
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Wei Zhang, Guang Yang, Steven P. Treon, Xiaofeng Zhang, Lian Xu, Xia Liu, Sara J. Buhrlage, Li Tan, Nathanael S. Gray, Jorge J. Castillo, Nicholas Tsakmaklis, Philip Cohen, Jiaji G. Chen, Jennifer R. Brown, Jie Chen, Christopher J. Patterson, Zachary R. Hunter, and Shuai Liu
- Subjects
0301 basic medicine ,Transcriptional Activation ,Proto-Oncogene Proteins c-hck ,Cell Survival ,Immunology ,Antineoplastic Agents ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Piperidines ,immune system diseases ,hemic and lymphatic diseases ,Cell Line, Tumor ,medicine ,Bruton's tyrosine kinase ,Humans ,Molecular Targeted Therapy ,Protein kinase A ,Protein kinase B ,biology ,Kinase ,Gene Expression Regulation, Leukemic ,Interleukin-6 ,Adenine ,Waldenstrom macroglobulinemia ,Cell Biology ,Hematology ,medicine.disease ,030104 developmental biology ,Pyrimidines ,chemistry ,030220 oncology & carcinogenesis ,Ibrutinib ,Myeloid Differentiation Factor 88 ,biology.protein ,Cancer research ,Pyrazoles ,Mutant Proteins ,Lymphoma, Large B-Cell, Diffuse ,Signal transduction ,Waldenstrom Macroglobulinemia - Abstract
Activating mutations in MYD88 are present in ∼95% of patients with Waldenstrom macroglobulinemia (WM), as well as other B-cell malignancies including activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL). In WM, mutated MYD88 triggers activation of Bruton tyrosine kinase (BTK). Ibrutinib, a pleiotropic kinase inhibitor that targets BTK, is highly active in patients with mutated MYD88. We observed that mutated MYD88 WM and ABC DLBCL cell lines, as well as primary WM cells show enhanced hematopoietic cell kinase (HCK) transcription and activation, and that HCK is activated by interleukin 6 (IL-6). Over-expression of mutated MYD88 triggers HCK and IL-6 transcription, whereas knockdown of HCK reduced survival and attenuated BTK, phosphoinositide 3-kinase/AKT, and mitogen-activated protein kinase/extracellular signal-regulated kinase signaling in mutated MYD88 WM and/or ABC DLBCL cells. Ibrutinib and the more potent HCK inhibitor A419259, blocked HCK activation and induced apoptosis in mutated MYD88 WM and ABC DLBCL cells. Docking and pull-down studies confirmed that HCK was a target of ibrutinib. Ibrutinib and A419259 also blocked adenosine triphosphate binding to HCK, whereas transduction of mutated MYD88 expressing WM cells with a mutated HCK gatekeeper greatly increased the half maximal effective concentration for ibrutinib and A419259. The findings support that HCK expression and activation is triggered by mutated MYD88, supports the growth and survival of mutated MYD88 WM and ABC DLBCL cells, and is a direct target of ibrutinib. HCK represents a novel target for therapeutic development in MYD88-mutated WM and ABC DLBCL, and possibly other diseases driven by mutated MYD88.
- Published
- 2016
37. Clonal architecture of CXCR4 WHIM-like mutations in Waldenström Macroglobulinaemia
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James L. Zehnder, Jennifer R. Brown, Yang Cao, Sandra Kanan, Yu-Tzu Tai, M. Lia Palomba, Alessandra Tedeschi, Jie Chen, Enrica Morra, Jorge J. Castillo, Xia Liu, Ruben D. Carrasco, Ranjana H. Advani, Marzia Varettoni, Nicholas Tsakmaklis, Lian Xu, Kimon V. Argyropoulos, Antonino Greco, Guang Yang, Zachary R. Hunter, Luca Arcaini, Nikhil M. Munshi, Alessandra Trojani, Kenneth C. Anderson, Jean Sabile, and Steven P. Treon
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Adult ,Male ,Receptors, CXCR4 ,Biology ,Compound heterozygosity ,medicine.disease_cause ,CXCR4 ,Monoclonal Gammopathy of Undetermined Significance ,Polymerase Chain Reaction ,Article ,Genomic Instability ,03 medical and health sciences ,symbols.namesake ,0302 clinical medicine ,medicine ,Humans ,Multiple myeloma ,Aged ,Sanger sequencing ,Aged, 80 and over ,Mutation ,Waldenstrom macroglobulinemia ,Hematology ,Lymphoma, B-Cell, Marginal Zone ,Sequence Analysis, DNA ,Middle Aged ,medicine.disease ,Molecular biology ,IgM Monoclonal Gammopathy ,Amino Acid Substitution ,Immunoglobulin M ,030220 oncology & carcinogenesis ,Immunology ,Myeloid Differentiation Factor 88 ,symbols ,Female ,Waldenstrom Macroglobulinemia ,Monoclonal gammopathy of undetermined significance ,030215 immunology - Abstract
CXCR4(WHIM) somatic mutations are distinctive to Waldenstrom Macroglobulinaemia (WM), and impact disease presentation and treatment outcome. The clonal architecture of CXCR4(WHIM) mutations remains to be delineated. We developed highly sensitive allele-specific polymerase chain reaction (AS-PCR) assays for detecting the most common CXCR4(WHIM) mutations (CXCR4(S338X C>A and C>G) ) in WM. The AS-PCR assays detected CXCR4(S338X) mutations in WM and IgM monoclonal gammopathy of unknown significance (MGUS) patients not revealed by Sanger sequencing. By combined AS-PCR and Sanger sequencing, CXCR4(WHIM) mutations were identified in 44/102 (43%), 21/62 (34%), 2/12 (17%) and 1/20 (5%) untreated WM, previously treated WM, IgM MGUS and marginal zone lymphoma patients, respectively, but no chronic lymphocytic leukaemia, multiple myeloma, non-IgM MGUS patients or healthy donors. Cancer cell fraction analysis in WM and IgM MGUS patients showed CXCR4(S338X) mutations were primarily subclonal, with highly variable clonal distribution (median 35·1%, range 1·2-97·5%). Combined AS-PCR and Sanger sequencing revealed multiple CXCR4(WHIM) mutations in many individual WM patients, including homozygous and compound heterozygous mutations validated by deep RNA sequencing. The findings show that CXCR4(WHIM) mutations are more common in WM than previously revealed, and are primarily subclonal, supporting their acquisition after MYD88(L265P) in WM oncogenesis. The presence of multiple CXCR4(WHIM) mutations within individual WM patients may be indicative of targeted CXCR4 genomic instability.
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- 2015
38. Mutated MYD88 Zygosity and CXCR4 Mutation Status Are Important Determinants of Ibrutinib Response and Progression Free Survival in Waldenstrom's Macroglobulinemia
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Xia Liu, Maria Demos, Christopher J. Patterson, Joshua Gustine, Jorge J. Castillo, Lian Xu, Maria Lia Palomba, Guang Yang, Nicholas Tsakmaklis, Ranjana H. Advani, Jiaji G. Chen, Steven P. Treon, Kirsten Meid, Toni Dubeau, Zachary R. Hunter, and Jie Chen
- Subjects
Mutation ,Immunology ,Macroglobulinemia ,Cell Biology ,Hematology ,Biology ,medicine.disease_cause ,Biochemistry ,Molecular biology ,Zygosity ,Frameshift mutation ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,chemistry ,030220 oncology & carcinogenesis ,Ibrutinib ,medicine ,Progression-free survival ,Allele ,Genotyping ,030215 immunology - Abstract
Activating somatic mutations in MYD88 and CXCR4 are present in 90-95% and 30-40% of untreated WM patients, respectively. Nearly all MYD88 somatic mutations involve a single nucleotide mutation that results in a change from leucine to proline at amino acid position 265, and most are heterozygous. In approximately 10-12% of untreated WM patients, MYD88 L265P is homozygous due to an acquired uniparenteral disomy (aUPD) or copy number alterations (Treon et al, NEJM 2012; Poulain et al, Blood 2013). Over 30 different types of nonsense and frameshift mutations in CXCR4 have been described in WM, and almost always are associated with mutated MYD88 (Hunter et al, Blood 2014). Mutated MYD88 activates BTK, and the BTK inhibitor ibrutinib is highly active in WM (Yang et al, Blood 2013; Treon et al NEJM 2015; Dimopoulos et al, ASH 2015). MYD88 mutations predict for response, while CXCR4 mutations are associated with slower response kinetics and lower response rates among mutated MYD88 WM patients on ibrutinib (Treon et al, NEJM 2015). In this study, we sought to clarify the impact of MYD88 homozygosity on ibrutinib activity in WM. We evaluated 33 previously treated, symptomatic WM patients who received ibrutinib on a clinical trial (NCT01614821), and for whom baseline and serial bone marrow (BM) CD19-selected cells were available for sequencing, copy number (CNA) and aUPD analysis. Mutated MYD88 homozygosity was determined by establishing the ratio of mutant (Mut) versus wild-type (WT) allele expression by Sanger sequencing. CNA status was determined using TaqMan real-time PCR assays. For patients with unaltered copy number, the presence of aUPD was determined by analyzing the tumor/germline allele balance using established TaqMan genotyping assays. At baseline, 9/33 (27.3%) of patients were homozygous for mutated MYD88. Mutated MYD88 homozygosity was confirmed to be due to deletion of the wild-type MYD88 allele by CNA in one patient, and amplification of the mutant MYD88 allele in another patient. TaqMan genotyping assays confirmed the presence of an aUPD in 6 patients that were confined to Chr. 3p. CXCR4 mutations were highly prevalent (8/9; 88.8%) among mutated MYD88 homozygous versus heterozygous (7/24; 29.2%) patients (p=0.014). We next assessed the impact of mutated MYD88 zygosity and CXCR4 mutation status on ibrutinib treatment outcome. The baseline characteristics for patients based on mutated MYD88 zygosity and CXCR4 mutation status are shown in Table 1. Following ibrutinib treatment, serum IgM levels declined by -0.89%, -0.54%, and -0.61% among MYD88Mut/WTCXCR4WT, MYD88Mut/WTCXCR4Mut, and MYD88Mut/Mut patients, respectively. Hemoglobin levels improved by 40.5%, 16.2% and 21.6% among MYD88Mut/WTCXCR4WT, MYD88Mut/WTCXCR4Mut, and MYD88Mut/Mut patients, respectively. Major responses (PR or better) were observed in 100%, 50%, and 89% of MYD88Mut/WTCXCR4WT, MYD88Mut/WTCXCR4Mut, and MYD88Mut/Mut patients, respectively. VGPR occurred in 50% of MYD88Mut/WTCXCR4WT patients, and 16.7% and 22.2% of MYD88Mut/WTCXCR4Mut, and MYD88Mut/Mut patients, respectively. Progression-free survival was also impacted by mutated MYD88 zygosity and CXCR4 mutation status (Figure 1). Median PFS was not reached for patients with MYD88Mut/WTCXCR4WT and MYD88Mut/Mut, and was 25.5 months for those with a MYD88Mut/WTCXCR4Mut mutation status (Log-rank Chi-square: 14.74, p=0.0006). Conclusions: MYD88 homozygosity is strongly associated with activating mutations in CXCR4, and appears to confer a better outcome for WM patients with CXCR4 mutations on ibrutinib therapy. Figure 1 Figure 1. Disclosures Treon: Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy. Castillo:Pharmacyclics: Honoraria; Abbvie: Research Funding; Otsuka: Consultancy; Biogen: Consultancy; Millennium: Research Funding; Janssen: Honoraria. Palomba:Pharmacyclics: Consultancy.
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- 2016
39. Molecular Basis of Ibrutinib Resistance in Waldenstrom's Macroglobulinemia
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Lian Xu, Steven P. Treon, Joshua Gustine, Jie Chen, Guang Yang, Toni Dubeau, Zachary R. Hunter, Christopher J. Patterson, Maria Demos, Xia Liu, Ranjana H. Advani, Jiaji G. Chen, Richard R. Furman, Kirsten Meid, Jorge J. Castillo, Maria Lia Palomba, and Nicholas Tsakmaklis
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0301 basic medicine ,Chronic lymphocytic leukemia ,Immunology ,medicine.disease_cause ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,hemic and lymphatic diseases ,medicine ,Bruton's tyrosine kinase ,Kinase activity ,Mutation ,biology ,business.industry ,Waldenstrom macroglobulinemia ,Macroglobulinemia ,Cell Biology ,Hematology ,medicine.disease ,030104 developmental biology ,chemistry ,Ibrutinib ,biology.protein ,Cancer research ,Mantle cell lymphoma ,business - Abstract
Ibrutinib is a small molecule that is approved by the U.S. FDA for the treatment of chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and Waldenström's Macroglobulinemia (WM). In WM, mutated MYD88 supports the growth and survival of malignant lymphoplasmacytic cells (LPC) through BTK, while CXCR4WHIM mutations promote ibrutinib resistance (Yang et al, Blood 2013; Cao et al, Leukemia 2013). Ibrutinib irreversibly binds to Cys481 on BTK, and blocks its kinase activity. Despite high response rates and durable remissions in WM (Treon et al, NEJM 2015; Dimopoulos et al, ASH 2015), disease progression can occur in WM patients on active ibrutinib therapy. To investigate the molecular basis of ibrutinib resistance in WM, we first focused on BTK mutations at Cys481 that have been associated with ibrutinib resistance in CLL and MCL using Sanger sequencing and nested AS-PCR. To capture the known variants at BTK Cys481, three AS-PCR assays for Cys481SerG>C, Cys481SerT>A, and Cys481ArgT>C were developed with a sensitivity of detecting 0.1% of mutant alleles. Using these assays, we evaluated 8 WM patients who progressed on ibrutinib. Among these 8 patients, 5 had BTK Cys481 mutations: 3 were positive for Cys481SerG>C, and2 were positive for all the three (Cys481SerG>C, Cys481SerT>A, and Cys481ArgT>C) mutations. Cloning/sequencing analysis confirmed co-occurrence of multiple Cys481 mutations within individual WM patients and the presence of mutations at different alleles. Furthermore, targeted deep sequencing (>300X coverage) confirmed all BTK Cys481 mutations, and identified an additional mutation at Cys481 (Cys481TyrG>A) in both patients who were positive for Cys481SerG>C, Cys481SerT>A and Cys481ArgT>C. The estimated allele frequencies by targeted deep sequencing for individual BTK mutations ranged from 1-34%. In contrast, no BTK Cys481 mutations were identified in 100 ibrutinib naive WM patients using the nested AS-PCR assays. Among the 8 WM patients included in this study, all had activating MYD88 mutations, and 4 had CXCR4WHIM mutations. All 4 patients with CXCR4WHIM mutations had BTK Cys481 mutations. We next utilized targeted deep sequencing to expand the mutation analysis to the entire coding regions of the BTK, as well as select genes relevant to BCR and MYD88 signaling. A missense mutation in CARD11 (L878F) was identified in one patient who lacked any BTK Cys481 mutations, while a missense mutation in PLCG2 (Y495H) was found in another patient with a Cys481SerG>C mutation. The findings provide the first reported insights into the molecular mechanisms associated with ibrutinib resistance in WM, and highlight the emergence of multiple BTK mutated clones within individual patients who progress on ibrutinib. Disclosures Castillo: Biogen: Consultancy; Abbvie: Research Funding; Pharmacyclics: Honoraria; Millennium: Research Funding; Otsuka: Consultancy; Janssen: Honoraria. Palomba:Pharmacyclics: Consultancy. Furman:Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Speakers Bureau. Treon:Janssen: Consultancy; Pharmacyclics: Consultancy, Research Funding.
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- 2016
40. Acquisition of BTK C481S Produces Resistance to Ibrutinib in MYD88 Mutated WM and ABC DLBCL Cells That Is Accompanied By ERK1/2 Hyperactivation, and Is Targeted By the Addition of the ERK1/2 Inhibitor Ulixertinib
- Author
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Nicholas Tsakmaklis, Guang Yang, Xia Liu, Christopher J. Patterson, Steven P. Treon, Maria Demos, Jorge J. Castillo, Lian Xu, Zachary R. Hunter, Jiaji G. Chen, and Jie Chen
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Immunology ,Biochemistry ,Viral vector ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,immune system diseases ,hemic and lymphatic diseases ,Ulixertinib ,Medicine ,Bruton's tyrosine kinase ,Protein kinase B ,biology ,business.industry ,Macroglobulinemia ,Cell Biology ,Hematology ,NFKB1 ,chemistry ,Cell culture ,030220 oncology & carcinogenesis ,Ibrutinib ,biology.protein ,Cancer research ,business ,030215 immunology - Abstract
Activating mutations in MYD88 mutations trigger BTK (Yang et al, Blood 2013; Wilson et al, Nat. Med. 2015) and HCK (Yang et al, Blood 2016) activation in Waldenström's macroglobulinemia (WM) and ABC-DLBCL, which activate multiple downstream pro-survival signaling cascades that include NFkB, AKT, and ERK1/2. Ibrutinib targets BTK and HCK, and shows high levels of activity in MYD88 mutated WM and ABC DLBCL. Resistance to ibrutinib has been observed in CLL, MCL, and in WM (Xu et al, submitted) due to acquisition of mutations that impact the binding of ibrutinib to BTK at Cysteine 481, which include the C481S mutation. To explore the functional impact of the BTK C481S mutation on ibrutinib treatment in MYD88 mutated WM and ABC DLBCL cells, we transduced MYD88 L265P mutated BCWM.1 and MWCL-1 WM, and TMD-8 and HBL-1 ABC DLBCL cell lines with either lentiviral vector alone, or lentiviral vectors expressing BTK wild-type (BTK WT) or BTK with C481S (BTK C481S) mutation. Transduction with BTK C481S led to a 1-3 log fold increase in EC50 for ibrutinib versus vector only or BTK WT transduced cells. BTK C481S expressing cells showed hyperphosphorylation of PLCγ2 (Tyr 1217) which is immediately downstream of BTK, and ERK1/2 (Thr 202/Tyr 204) versus vector only or BTK WT cells in all four MYD88 mutated cell lines following ibrutinib (0.1, 0.5 uM) treatment for 2 hrs. In contrast, no changes in the phospho-IKBa (Ser 32), the gatekeeper of NFkB, nor phospho-AKT (Ser 473) or p38MAPK (Thr 180/Tyr 182) were observed between vector only, BTK WT or BTK C481S transduced cells following ibrutinib treatment. Given the hyperactivation of ERK1/2 in BTK C481S transduced cells treated with ibrutinib, we next evaluated the activity of ulixertinib (BVD-523, VRT752271), a highly selective ERK1/2 inhibitor that is currently under clinical investigation. Ulixertinib blocked ERK1/2 activity as evidenced by reduction of phospho-p90RSK (Ser 380) by western blot analysis in BTK C481S transduced BCWM.1 and TMD-8 cells. The combination of ulixertinib with ibrutinib produced higher levels of tumor cell killing than either agent alone, and showed synergistic interactions with combination index (CI) values below 1.0 at pharmacologically relevant concentrations. Our data indicate that acquisition of BTK C481S produces resistance to ibrutinib in MYD88 mutated WM and ABC DLBCL cells that is accompanied by ERK1/2 hyperactivation. The addition of the ERK1/2 inhibitor ulixertinib to ibrutinib at pharmacologically relevant concentrations produced synergistic tumor cell killing in BTK C481S ibrutinib resistant cells. The findings provide rationale for the investigation of ERK1/2 inhibitors in ibrutinib resistant MYD88 driven WM and ABC DLBCL disease mediated by BTK mutations. Disclosures Castillo: Pharmacyclics: Honoraria; Janssen: Honoraria; Millennium: Research Funding; Biogen: Consultancy; Otsuka: Consultancy; Abbvie: Research Funding. Treon:Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy.
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- 2016
41. HCK Transcription Is Regulated By AP1, NF-Kb and STAT3 Transcription Factors in MYD88 Mutated WM and ABC-DLBCL Cells
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Christopher J. Patterson, Xia Liu, Nicholas Tsakmaklis, Lian Xu, Jie Chen, Jorge J. Castillo, Maria Demos, Steven P. Treon, Guang Yang, Jiaji G. Chen, and Zachary R. Hunter
- Subjects
JUNB ,Immunology ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Molecular biology ,AP-1 transcription factor ,Transcription (biology) ,Cell culture ,Gene expression ,biology.protein ,STAT3 ,Transcription factor ,Tyrosine kinase - Abstract
Hematopoietic cell kinase (HCK) is a member of the SRC family of tyrosine kinases (SFKs). HCK transcription is aberrantly upregulated in Waldenström's Macroglobulinemia (WM) and Activated B-cell (ABC) subtype Diffuse Large B-cell Lymphoma (DLBCL) in response to activating mutations in MYD88 (Yang et al, Blood 2016). To clarify the mechanism responsible for the aberrant upregulation of HCK transcription inMYD88 mutated cells, we analyzed the promoter sequence of HCK using PROMO and identified consensus binding sites for transcription factors (AP1, NF-kB, STAT3, and IRF1) that are regulated by mutated MYD88 (Ngo et al, Nature 2011; Treon et al, NEJM 2012; Yang et al, Blood 2013; Juilland et al, Blood 2016; Yang et al, Blood 2016). We performed Chromatin Immuno-precipitation (ChIP) assays using ChIP grade antibodies to JunB, c-Jun, NF-kB-p65, STAT3 and IRF1 in MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8, HBL-1, OCI-Ly3) cells that highly express HCK transcripts, as well as wild type MYD88 expressing GCB DLBCL (OCI-Ly7, OCI-Ly19) cells that show low HCK transcription. Following ChIP, a HCK promoter specific quantitative PCR assay was used to detect HCK promoter sequences. These studies showed that JunB, NF-kB-p65 and STAT3 bound more robustly to the HCK promoter in MYD88 mutated WM and ABC-DLBCL cells versus MYD88 wild type GCB DLBCL cell lines, while c-Jun bound more abundantly to the HCK promoter sequence in all DLBCL cell lines, regardless of MYD88 mutation status. In contrast c-Jun binding was low in MYD88 mutated WM cells. IRF1 binding to the HCK promoter was similar in all cell lines, regardless of the MYD88 mutation status. To further investigate HCK regulation, we developed an HCK promoter driven luciferase reporter vector (WT) with mutated AP-1 binding (AP1-mu-1~6), NF-kB binding (NF-kB-mu-1~5), and STAT3 binding (STAT3-mu) sites and investigated their impact on HCK promoter activity in MYD88 mutated BCWM.1 cells. We observed that mutation of AP1-mu-1,4,5,6; NF-kB-mu-1,4,5, as well as STAT3-mu binding sites greatly reduced HCK promoter activity, thereby supporting a role for AP-1, NF-kB and STAT3 transcription factors in HCK gene expression in MYD88 mutated cells. To further clarify the importance of these transcription factors in aberrant HCK gene expression in MYD88 mutated cells, we treated BCWM.1, MWCL-1, TMD-8 and HBL-1 cells with the AP-1 inhibitor SR 11302; NF-kB inhibitor QNZ; and the STAT3 inhibitor STA-21. Treatment of cells for 2 hours with SR 11302, QNZ, and STA-21 at sub-EC50 concentrations resulted in decreased HCK expression in MYD88 mutated all cell lines. Lastly, we investigated the contribution of BCR signaling to HCK transcription. BCWM.1, MWCL-1, TMD-8, and HBL-1 cells were treated with the Syk kinase inhibitor R406, and HCK transcription levels were then assessed. Differences in HCK expression were observed between MYD88 mutated WM and ABC DLBCL cells following R406, supporting a contributing role for BCR signaling in ABC DLBCL but not WM cells to HCK expression. Our data provide critical new insights into HCK regulation, and a framework for targeting pro-survival HCK signaling in WM and ABC DLBCL cells dependent on activating MYD88 mutations. Disclosures Castillo: Biogen: Consultancy; Otsuka: Consultancy; Millennium: Research Funding; Janssen: Honoraria; Abbvie: Research Funding; Pharmacyclics: Honoraria. Treon:Janssen: Consultancy; Pharmacyclics: Consultancy, Research Funding.
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- 2016
42. Targeting Myddosome Self-Assembly in Waldenstrom's Macroglobulinemia
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Nicholas Tsakmaklis, Jie Chen, Xia Liu, Nathanael S. Gray, Guang Yang, Sara J. Buhrlage, Jorge J. Castillo, Lian Xu, Steven P. Treon, Jiaji Chen, Zachary R. Hunter, and Christopher J. Patterson
- Subjects
biology ,Cell growth ,Kinase ,Immunology ,Caspase 3 ,Cell Biology ,Hematology ,IRAK4 ,Biochemistry ,Transduction (genetics) ,Annexin ,biology.protein ,Cancer research ,Bruton's tyrosine kinase ,Death domain - Abstract
Background: MYD88 mutations are present in over 95% of patients with Waldenstrom's Macroglobulinemia (WM), and promote Myddosome self-assembly that triggers NFκB-dependent survival through BTK and IRAK1/IRAK4 (Blood 122(7):1222-32). While current therapeutic strategies are aimed at blocking these downstream kinases, peptidomimetics that interfere with Myddosome self-assembly may offer a more targeted approach for blocking aberrant MYD88 signaling. Methods: We expressed by lentiviral transduction mini-peptides of MYD88 Toll/Interleukin-1 Receptor (TIR) or Death Domain (DD) sequences in mutated MYD88 WM and wild-type MYD88 control cells (Figure 1). We used phospho-flow analysis to evaluate for changes in pBTK, pIRAK1/IRAK4, and pNFKB, and determined cell growth and survival by Alamar Blue Assay, Annexin V, and cleaved Caspase 3 staining. Results: Transduction of TIR or DD mini-peptides in mutated MYD88 WM cells but not wild-type MYD88 control cells reduced NFKB activation and tumor cell growth, and prompted Annexin V and/or cleaved Caspase 3 staining. TIR interfering mini-peptides impacted BTK but not IRAK1/IRAK4 activation, whereas DD interfering mini-peptides showed an opposite effect. Conclusions: The findings demonstrate differences in BTK versus IRAK1/IRAK4 directed NFKB signaling in response to Myddosome self-assembly in MYD88 mutated WM cells. The feasibility of directly targeting MYD88 homodimerization to block aberrant MYD88 signaling was also recognized, and suitable peptide sequences for the development of peptidomimetics that interfere with Myddosome self-assembly and signaling were identified. The findings provide a framework for direct targeting of the Myddosome in MYD88 mutated WM disease. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.
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- 2015
43. Whole Genome and Transcriptome Analysis of Waldenstrom's Cell Lines Show Preservation of Many Important Genomic Features of Primary Disease, and Identify a Non-L265P Mutation in MYD88
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Christopher J. Patterson, Yang Cao, Lian Xu, Jiaji Chen, Zachary R. Hunter, Xia Liu, Steven P. Treon, Guang Yang, Jie Chen, Jorge J. Castillo, and Nicholas Tsakmaklis
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Genetics ,Immunology ,Genomics ,Cell Biology ,Hematology ,CD38 ,Biology ,Biochemistry ,Genome ,DNA sequencing ,Germline ,Transcriptome ,Gene expression profiling ,Chromosome 3 - Abstract
Background: WM cell lines represent a vital tool for discovery of mechanisms responsible for WM pathogenesis, cell signaling, development of targeted therapeutics, and evaluation of mechanisms responsible for drug resistance. To date, there has been no comprehensive genomic and transcriptomic profiling of WM cell lines using next generation sequencing. Methods: We performed whole genome (WGS) and transcriptome sequencing (WTS) on three established (BCWM.1, MWCL-1, RPCI-WM1), and one novel (BCWM.2) cell line established from continuous culture of CD19-selected bone marrow cells from a WM patient with an immunophenotype consistent with primary WM disease (CD5-, CD10-, CD19+, CD20+, sIgM+, λ+, CD11c-, CD38+, and CD138+). Samples from primary tumor and germline from the founder of BCWM.2, along with tumor samples from 57 WM patients, as well as memory (CD19+ CD27+) and non-memory (CD19+ CD27-) B-cells from 9 healthy donors were also sequenced. WGS was performed using Complete Genomics platform, and WTS using Illumina HiSeq. Reads were aligned to KnownGene HG19/GRCh37 reference using STAR. Read counts per gene were obtained using featureCounts from Rsubread, and analyzed using voom from the edgeR/limma Bioconductor packages in R. Differential isoform expression was assessed using the Cufflinks software suite. Results: WGS revealed no cytogenetic level amplifications or deletions in BCWM.1, whereas MWCL-1 harbored monoallelic deletions in 17p and 17q, and amplifications in 11q. RPCI-WM1 showed deletions in 3p, 6q, 9p, 13q, 18q, 19q and X, with amplifications in 5p, 6p, 7q, 11q, 14q, 18q, 21q, and acquired uniparental disomies on 17p, 18q, and X. BCWM.2 matched its parent tumor profile with trisomy 3 and 12, as well as deletion of the entire arm of 6q and corresponding amplification of 6p. All 4 cell lines were wild type for CXCR4 and all but BCWM.2 harbored the MYD88 L265P mutation. Like its founding clone, BCWM.2 carried an activating heterozygous MYD88 G/A S243N mutation (Nature 470(7332):115-9) previously described in ABC DLBCL. In addition, a novel I318N mutation in LYN that was predicted to be activating was found in BCWM.2, but not in the founder primary tumor or germline samples. While WTS analysis of RPCI-WM1 is pending, principal component analysis showed that BCWM.1, BCWM.2 and MWCL-1 cells clustered with primary WM tumor cells, and were distinct from memory and non-memory B-cell samples from healthy donors. Likewise, multidimensional scaling of pairwise similarity clustered BCWM.2 directly with its corresponding primary sample (Figure 1). BCWM.1, BCWM.2, and MWCL-1 cells showed a high degree of recapitulation with primary MYD88mutated CXCR4wild-type WM cells including IL17RB, CABLES1, WNT5A, GPER, or WNK2 over-expression. As a group, WM cell lines were found to over-express many cancer associated genes, including MUC13, MMP7, LMO3, IL4I1, and several SRC family kinases. Conclusions: This study provides the first ever comprehensive genomic and transcriptomic profiling of WM cell lines by next generation sequencing. The findings show preservation of many important genomic features of primary WM disease, and identified the existence of a non-L265P activating MYD88 mutation, as well as dysregulation of many cancer associated genes in WM. The findings provide an important new resource for the study of WM disease. Disclosures No relevant conflicts of interest to declare.
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- 2015
44. Targeting IRAK1/IRAK4 Signaling in Waldenstrom's Macroglobulinemia
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Xia Liu, Jie Chen, Jorge J. Castillo, Lian Xu, Christopher J. Patterson, Nathanael S. Gray, Philip Cohen, Steven P. Treon, Nicholas Tsakmaklis, Sara J. Buhrlage, Jiaji Chen, Li Tan, and Guang Yang
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Gene knockdown ,biology ,Immunology ,Waldenstrom macroglobulinemia ,Cell Biology ,Hematology ,IRAK4 ,medicine.disease ,Biochemistry ,chemistry.chemical_compound ,Cell killing ,chemistry ,Apoptosis ,Ibrutinib ,biology.protein ,Cancer research ,medicine ,Bruton's tyrosine kinase ,Viability assay - Abstract
Background: MYD88 L265P somatic mutations are highly prevalent in Waldenström's macroglobulinemia (WM) (NEJM 367(9):826-33). MYD88 L265P activates multiple downstream signaling pathways including BTK and IRAK1/IRAK4 that support malignant cell growth and survival (Nature 470(7332):115-9; Blood 122(7):1222-32). Ibrutinib targets BTK, and shows high overall and major clinical response rates, though no complete responses are observed indicating alternative survival signaling. Methodology: Phospho-flow analysis of IRAK1, IRAK4, and BTK was performed in primary WM cells taken from untreated WM patients, and those on ibrutinib therapy. IRAK1 or IRAK4 knockdown experiments were performed using lentiviral shRNA transduction. Immunoprecipitation, western blot and phospho-flow studies were used to detect protein expression and phosphorylation in WM cells. Cell survival following IRAK4 or IRAK1 knockdown, ibrutinib and/or IRAK4/IRAK1 inhibitor (EMD Millipore) treatment was assessed by Annexin V staining, AlamarBlue® Cell Viability Assay or CellTiter-Glo® Luminescent Cell Viability Assays. Results: Phospho-flow analysis of bone marrow lymphoplasmacytic cells taken from WM patients following > 6 months of continued ibrutinib treatment demonstrated highly active IRAK1 and IRAK4, but not BTK. These findings prompted us to dissect the relative impact of IRAK1 and IRAK4 in supporting WM cell survival. Using lentiviral transduction, we identified shRNAs that produced similar levels of protein reduction by western blot analysis for both IRAK1 and IRAK4. Compared to scrambled control vector, knockdown of IRAK1 or IRAK4 both produced decreased tumor cell survival in MYD88 mutated BCWM.1 and MWCL-1 cells. More pronounced apoptosis, as well as sustained reduction in tumor cell growth occurred following knockdown of IRAK1 versus IRAK4. Treatment of primary WM cells taken from untreated patients, patients on ibrutinib therapy, as well as MYD88 mutated WM cells lines with ibrutinib and a toolbox IRAK4/IRAK1 inhibitor resulted in more robust reductions in NFkB signaling, and at least additive tumor cell killing versus either agent alone. Conclusions: MYD88 L265P mutated WM cells show greater dependence on IRAK1 versus IRAK4 directed signaling. IRAK1/IRAK4 signaling may contribute to persistent WM cell survival following ibrutinib treatment. Combined BTK and IRAK inhibition leads to augmented blockade of NFKB signaling and enhanced WM cell killing. The studies provide a framework for the development and investigation of IRAK inhibitors, alone and in combination with ibrutinib in WM patients. Disclosures No relevant conflicts of interest to declare.
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- 2015
45. The Clonal Architecture of CXCR4mutations in Waldenstrom's Macroglobulinemia Shows Highly Variable Subclonal Distribution, and Multiple Mutations within Individual Patients Indicative of Targeted Genomic Instability
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Lian Xu, Nikhil C. Munshi, Kenneth C. Anderson, Jorge J. Castillo, Steven P. Treon, Marzia Varettoni, Nicholas Tsakmaklis, James L. Zehnder, Xia Liu, Ruben D. Carrasco, Enrica Morra, Ranjana H. Advani, Kimon V. Argyropoulos, M. Lia Palomba, Guang Yang, Alessandra Tedeschi, Zachary R. Hunter, Jennifer R. Brown, Antonino Greco, Luca Arcaini, Jie Chen, Sandra Kanan, Yu-Tzu Tai, Alessandra Trojani, and Yang Cao
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Sanger sequencing ,Genetics ,Whole genome sequencing ,Mutation ,Immunology ,Waldenstrom macroglobulinemia ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Compound heterozygosity ,medicine.disease_cause ,Biochemistry ,Molecular biology ,DNA sequencing ,Frameshift mutation ,symbols.namesake ,medicine ,symbols ,Monoclonal gammopathy of undetermined significance - Abstract
Background: Whole genome sequencing (WGS) identified activating CXCR4WHIM somatic mutations in nearly 30% of patients with Waldenstrom's Macroglobulinemia (WM) (Blood 123(11):1637-46). Both nonsense and frameshift CXCR4WHIM mutations occur in WM, with over 30 different types of mutations described within the regulatory carboxyl-terminal domain of CXCR4. CXCR4WHIM mutations almost always occur with activating MYD88 mutations, and impact both disease presentation and treatment outcome (Blood 123(18):2791-6; NEJM 372(15):1430-40.). The clonal architecture of CXCR4WHIM mutations relative to MYD88 mutations and their role in disease evolution remains to be clarified. Methods: We used Sanger sequencing and highly sensitive AS-PCR assays that we developed for the most common CXCR4WHIM mutations (S338X C>A and C>G) to evaluate for CXCR4WHIM mutations. In conjunction with an AS-PCR MYD88L265P assay that we previously developed (Leukemia 28(8):1698-707), we also profiled tumor samples for MYD88L265P and CXCR4S338X mutations in 164 WM, 12 IgM MGUS, 20 MZL, 32 CLL, 14 MM, 7 non-IGM MGUS patients, and 32 healthy donors. Next generation transcriptome sequencing data was also performed for validation in select cases. Results: AS-PCR detected CXCR4S338X mutations in WM and IgM MGUS patients not revealed by Sanger sequencing. By combined AS-PCR and Sanger sequencing, CXCR4WHIM mutations were identified in 44/102 (43%), 21/62 (34%), 2/12 (17%), and 1/20 (5%) untreated WM, previously treated WM, IgM MGUS and MZL patients, respectively, but not in CLL, MM, non-IGM MGUS patients or healthy donors. Cancer cell fraction analysis in WM and IgM MGUS patients showed CXCR4S338X mutations were primarily subclonal, with highly variable clonal distribution (median 35.1%, range 1.2%-97.5%; Figure 1). Sanger sequencing identified 3 patients with multiple CXCR4 mutations, which were shown to be compound heterozygous by TA cloning and sequencing of at least 40 clones. The addition of AS-PCR to the Sanger sequencing results also revealed multiple CXCR4WHIM mutations in many individual patients that included homozygous and compound heterozygous mutations that were validated by next generation sequencing that offered a median of 5,819 (range 5,217-12,235) reads that overlapped the mutated loci. Conclusions: Taken together, these findings show that CXCR4WHIM mutations are more common in WM patients than previously revealed by WGS or Sanger sequencing. Moreover, CXCR4 mutations are primarily subclonal supporting their acquisition after MYD88L265P in WM oncogenesis. The exclusive finding of frameshift and nonsense but not missense variants within the carboxyl-terminal domain of CXCR4 suggests that significant selection pressures exist for activating mutations within the WM clone. Lastly, multiple CXCR4WHIM mutations are common in WM patients indicative of targeted genomic instability within the carboxyl-terminal domain of CXCR4. Disclosures No relevant conflicts of interest to declare.
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- 2015
46. Next Generation Sequencing Identifies a Distinct Transcriptional Profile, Including Isoform Dysregulation That Segue with Genomic Alterations in Waldenstrom's Macoglobulinemia
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Christopher J. Patterson, Kenneth C. Anderson, Xia Liu, Guang Yang, Steven P. Treon, Robert R Manning, Nicholas Tsakmaklis, Lian Xu, Jie Chen, Nikhil C. Munshi, Jorge J. Castillo, Zachary R. Hunter, and Jiaji Chen
- Subjects
Whole genome sequencing ,Gene isoform ,Genetics ,Sanger sequencing ,Mutation ,Immunology ,Cell Biology ,Hematology ,Biology ,medicine.disease_cause ,Biochemistry ,DNA sequencing ,symbols.namesake ,Gene expression ,PRDM1 ,medicine ,symbols ,Gene - Abstract
Background: Whole genome sequencing has identified highly prevalent somatic mutations including MYD88, CXCR4 and ARID1A, as well as gene losses in Waldenström's Macroglobulinemia (WM) (NEJM 367(9):826-33; Blood 123(11):1637-46). At least three genomic defined subpopulations of WM have been identified based on MYD88 and CXCR4 mutation status (Blood 123(18):2791-6).The regulatory impact for these genomic alterations remain to be clarified. Methods: Next generation transcriptome profiling was performed using Illumina HiSeq. RNA was isolated from CD19-selected bone marrow cells from 57 WM patients, as well as memory (CD19+CD27+) and non-memory (CD19+CD27-) B-cells from 9 healthy donors. Differential gene expression analysis was performed based on MYD88, CXCR4 and ARID1A mutation status, as well as common cytogenetic abnormalities encountered in WM including amplifications in chromosomes 3q, 4, 6p, and deletions in 6q. Reads were aligned to KnownGene HG19/GRCh37 reference using STAR. Read counts per gene were obtained using featureCounts from Rsubread, and analyzed using voom from the edgeR/limma Bioconductor packages in R. Differential isoform expression was assessed using the Cufflinks software suite. Functional enrichment analysis was conducted using Ingenuity Pathway Analysis. Results: Transcriptional profilingof WM cells showed a stronger correlation with healthy donor memory B-cells, their putative cell of origin. Differential gene expression analysis between WM patients and healthy donors generated a distinct transcriptional profile for WM that included dysregulation of PIM1, RGS7, IL11RA and EPHB4, as well as differential isoform usage in TP53, PRDM1 and XBP1. MYD88mutatedCXCR4wild-type(WT) WM patients consistently over-expressed genes unique to this cohort including, IL17RB, GPER, WNT5A, WNK2, CABELS1, and PRDM5. MYD88mutated patients who harbored nonsense (NS) or frame-shift (FS) mutations in CXCR4 showed functional enrichment of genes that indicated inhibition of Toll-like receptor inflammatory pathways. CXCR4 mutated patients showed strong up-regulation of CXCR7 and TSPAN33, and exhibited isoform level dysregulation of MEF2B, FOXO3, KDM2A and PRKAG2. Unsupervised clustering differentiated MYD88mutatedCXCR4WT from MYD88mutatedCXCR4NS/FS patients, while samples from MYD88WTCXCR4WT clustered with CXCR4NS/FS group. Clusters based on clonality and disease burden were also observed (Figure 1). CXCR4NS/FS transcripts were preferentially expressed over CXCR4WT transcripts despite the subclonal presence of CXCR4mutations. These findings were further validated by comparative DNA versus cDNA Sanger sequencing indicating allelic dysregulation within CXCR4NS/FS subclones. Controlling for MYD88 and CXCR4 mutation status, the presence of ARID1A mutations and cytogenetic abnormalities generated distinct transcriptional profiles. Random forest regression analysis identified subsets of genes strongly associated with bone marrow disease involvement, serum IgM and hemoglobin levels. Notably, increased bone marrow disease burden was associated with increased CXCL13, and decreased TP53 and RBL1 expression. Likewise, higher levels of serum IgM corresponded with increased IL27RA expression. Conclusion: Using next generation sequencing, we have identified a distinct transcriptional profile, including isoform dysregulation that segue with highly prevalent genomic mutations in WM. The findings provide valuable insights into the molecular pathogenesis and clinical presentation of WM. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.
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- 2015
47. HCK Is a Highly Relevant Target of Ibrutinib in MYD88 Mutated Waldenstrom's Macroglobulinemia and Diffuse Large B-Cell Lymphoma
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Sara J. Buhrlage, Jiaji Chen, Li Tan, Jorge J. Castillo, Wei Zhang, Nicholas Tsakmaklis, Xia Liu, Zachary R. Hunter, Christopher J. Patterson, Jie Chen, Steven P. Treon, Nathanael S. Gray, Guang Yang, and Lian Xu
- Subjects
biology ,Kinase ,Immunology ,Waldenstrom macroglobulinemia ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,CD19 ,chemistry.chemical_compound ,chemistry ,Cell culture ,Ibrutinib ,biology.protein ,medicine ,Cancer research ,Bruton's tyrosine kinase ,Kinome ,Diffuse large B-cell lymphoma - Abstract
Background: Ibrutinib was recently approved by the U.S. FDA, and the European Medicines Agency for the treatment of Waldenström's macroglobulinemia (WM) given the findings of a prospective, multicenter study that showed high levels of response and durable progression-free survival in previously treated patients (NEJM 372(15):1430-40). Mutations in MYD88 are highly prevalent in patients with WM (NEJM 367(9):826-33) and ABC subtype of DLBCL (Nature 470(7332):115-9), and activate BTK, a target of ibrutinib (Blood 122(7):1222-32). Ibrutinib is also known to target other kinases, which may be functionally relevant to its activity in WM, and other MYD88 mutated diseases. Methodology: Transcriptomic profiling of primary WM patient cells; MYD88, HCK and BTK transduction experiments; Kinome profiling; and co-immunoprecipitation studies using biotin-labeled ibrutinib were performed to identify relevant targets of ibrutinib in MYD88 mutated cells. Functional studies were also performed to validate target activity. Results: Transcriptomic profiling and quantitative RT-PCR validation studies identified aberrant over-expression of HCK in bone marrow derived malignant cells (CD19+) from WM patients with MYD88 mutations versus memory (CD19+ CD27+) and non-memory (CD19+ CD27-) B-cells from healthy donors. HCK was highly expressed in MYD88 mutated WM and ABC-type DLBCL cell lines (BCWM.1, MWCL-1, TMD-8, HBL-1, OCI-Ly3) versus MYD88 wild type cell lines (OCI-Ly7, OCI-Ly19, Ramos, MM1.S and RPMI-8226). Over-expression of MYD88 L265P but not wild-type MYD88 resulted in significantly higher levels of HCK, while knockdown of HCK and/or use of a known HCK inhibitor (A419259) reduced survival of MYD88 mutated WM and DLBCL cell lines, and primary WM cells. Importantly, HCK (Tyr411) was highly phosphorylated in primary WM cells, as well as WM and DLBCL cell lines expressing the MYD88 L265P mutation, and its level of activation was enhanced by treatment with IL-6, a major growth and survival factor. Knockdown of the IL-6 co-receptor GP-130 abrogated IL-6 triggered activation of HCK. Kinome screening showed that HCK was a target of ibrutinib, while biotin labeled ibrutinib but not AVL-292 (CC-292) showed robust binding to HCK in BCWM.1, MWCL-1 and TMD-8 cells. Furthermore, by use of a kinase active-site inhibition assay, ibrutinib but not AVL-292 blocked ATP binding to HCK in a dose-dependent manner, while both compounds blocked ATP binding to BTK. To elaborate on the importance of HCK inhibition by ibrutinib in WM growth and survival, WM cells were transduced with vectors expressing a HCK gatekeeper mutant (HCK-T338M) or an ibrutinib BTK binding site mutant (BTK-C481S). Transduction with HCK-T338M significantly increased the EC50 for ibrutinib or A419259 in comparison to wild type HCK expressing BCWM.1 cells, while no rescue effect was observed for AVL-292 treated cells. Transduction with BTK-C481S showed rescue for ibrutinib and AVL-292, but not A419259. By phospho-flow analysis, ibrutinib and A419259, but not AVL-292 significantly reduced HCK Tyr411 phosphorylation in MYD88 L265P expressing WM and DLBCL cell lines. Conclusions: HCK is regulated by MYD88 L265P, and is activated in primary WM cells, as well as MYD88 L265P expressing WM and DLBCL cell lines. IL-6 activates HCK though GP130, and inhibition of HCK abrogates WM cell survival. Ibrutinib shows robust binding to HCK, and transduction of WM cells with a mutated HCK gatekeeper blocks ibrutinib related tumor cell killing. These studies identify HCK as a novel, and highly relevant target of ibrutinib in MYD88 mutated WM and DLBCL cells. Disclosures No relevant conflicts of interest to declare.
- Published
- 2015
48. Ibrutinib Monotherapy in Symptomatic, Treatment-Naïve Patients With Waldenström Macroglobulinemia.
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Treon SP, Gustine J, Meid K, Yang G, Xu L, Liu X, Demos M, Kofides A, Tsakmaklis N, Chen JG, Munshi M, Chan G, Dubeau T, Raje N, Yee A, O'Donnell E, Hunter ZR, and Castillo JJ
- Subjects
- Adenine analogs & derivatives, Adult, Aged, Aged, 80 and over, Female, Humans, Male, Middle Aged, Mutation, Myeloid Differentiation Factor 88 genetics, Piperidines, Receptors, CXCR4 genetics, Waldenstrom Macroglobulinemia genetics, Antineoplastic Agents therapeutic use, Pyrazoles therapeutic use, Pyrimidines therapeutic use, Waldenstrom Macroglobulinemia drug therapy
- Abstract
Purpose Ibrutinib is active in previously treated Waldenström macroglobulinemia (WM). MYD88 mutations ( MYD88
MUT ) and CXCR4 mutations ( CXCR4MUT ) affect ibrutinib response. We report on a prospective study of ibrutinib monotherapy in symptomatic, untreated patients with WM, and the effect of CXCR4MUT status on outcome. Patients and Methods Symptomatic, treatment-naïve patients with WM were eligible. Ibrutinib (420 mg) was administered daily until progression or unacceptable toxicity. All tumors were genotyped for MYD88MUT and CXCR4MUT . Results A total of 30 patients with WM received ibrutinib. All carried MYD88MUT , and 14 (47%) carried a CXCR4MUT . After ibrutinib treatment, median serum IgM levels declined from 4,370 to 1,513 mg/dL, bone marrow involvement declined from 65% to 20%, and hemoglobin level rose from 10.3 to 13.9 g/dL ( P < .001 for all comparisons). Overall (minor or more than minor) and major (partial or greater than partial) responses for all patients were 100% and 83%, respectively. Rates of major (94% v 71%) and very good partial (31 v 7%) responses were higher and time to major responses more rapid (1.8 v 7.3 months; P = 0.01) in patients with wild-type CXCR4 versus those with CXCR4MUT , respectively. With a median follow-up of 14.6 months, disease in two patients (both with CXCR4MUT ) progressed. The 18-month, estimated progression-free survival is 92% (95% CI, 73% to 98%). All patients are alive. Grade 2/3 treatment-related toxicities in > 5% of patients included arthralgia (7%), bruising (7%), neutropenia (7%), upper respiratory tract infection (7%), urinary tract infection (7%), atrial fibrillation (10%), and hypertension (13%). There were no grade 4 or unexpected toxicities. Conclusion Ibrutinib is highly active, produces durable responses, and is safe as primary therapy in patients with symptomatic WM. CXCR4MUT status affects responses to ibrutinib.- Published
- 2018
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