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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.

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.

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.

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.

Key Points Single-cell whole-genome amplification can be used to interrogate the genomic architecture of Waldenström’s macroglobulinemia. The mutational signature of CXCR4MUT cells may be associated with alterations in DNA repairing genes and tumor suppressors.

Treon et al provide early clinical data supporting a theoretical rationale for continuing ibrutinib in patients receiving the drug during COVID-19 illness.

Hematopoietic cell kinase (HCK) is an SRC family member that is aberrantly upregulated in B-cell neoplasms dependent on MYD88-activating mutations and supports their growth and survival. We showed herein that activation of Toll-like receptor (TLR) signaling in MYD88 wild-type B cells also triggered HCK expression, denoting on path regulatory function for HCK by MYD88. To clarify the signaling cascades responsible for aberrant HCK expression in MYD88-mutated B-cell lymphomas, we performed promoter-binding transcription factor (TF) profiling, PROMO weighted TF consensus binding motif analysis, and chromatin immunoprecipitation studies. We identified PAX5, and the mutated MYD88 downstream signaling mediators STAT3, NF-κB, and AP-1, 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. Among AP-1 complex components, JunB showed greatest relevance to TLR/MYD88 signaling and HCK transcription regulation. In MYD88-mutated Waldenström macroglobulinemia and activated B-cell-diffuse large B-cell lymphoma cells, knockdown of MYD88 reduced phosphorylation of JunB but not c-Jun, and knockdown of JunB reduced HCK protein levels. Deletion of STAT3, NF-κB, and AP-1 binding sites reduced corresponding TFs binding and HCK promoter activity. Moreover, inhibitors to STAT3, NF-κB, and AP-1 reduced HCK promoter activity and messenger RNA levels, particularly in combination, in MYD88-mutated lymphoma cells. The findings provide new insights into the transcriptional regulation of HCK prosurvival signaling by mutated MYD88, and the importance of JunB as a downstream mediator of the MYD88-directed signaling apparatus.

MYD88 L265P is the most common mutation in lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) and one of the most frequent in poor-prognosis subtypes of diffuse large B-cell lymphoma (DLBCL). Although inhibition of the mutated MYD88 pathway has an adverse impact on LPL/WM and DLBCL cell survival, its role in lymphoma initiation remains to be clarified. We show that in mice, human MYD88L265P promotes development of a non-clonal, low-grade B-cell lymphoproliferative disorder with several clinicopathologic features that resemble human LPL/WM, including expansion of lymphoplasmacytoid cells, increased serum immunoglobulin M (IgM) concentration, rouleaux formation, increased number of mast cells in the bone marrow, and proinflammatory signaling that progresses sporadically to clonal, high-grade DLBCL. Murine findings regarding differences in the pattern of MYD88 staining and immune infiltrates in the bone marrows of MYD88 wild-type (MYD88WT) and MYD88L265P mice are recapitulated in the human setting, which provides insight into LPL/WM pathogenesis. Furthermore, histologic transformation to DLBCL is associated with acquisition of secondary genetic lesions frequently seen in de novo human DLBCL as well as LPL/WM-transformed cases. These findings indicate that, although the MYD88L265P mutation might be indispensable for the LPL/WM phenotype, it is insufficient by itself to drive malignant transformation in B cells and relies on other, potentially targetable cooperating genetic events for full development of lymphoma.

The SRC family kinase (SFK) HCK is transcriptionally upregulated and activated by mutated MYD88 (MYD88Mut), a key adaptor for Toll-receptor signaling. HCK activates BTK, AKT, and ERK in MYD88Mut lymphomas. SYK, a B-cell receptor (BCR) component, is activated in MYD88Mut lymphoma cells. Although the SFK LYN serves as a trigger for SYK activation in MYD88Mut ABC DLBCL cells, LYN activity is muted in MYD88Mut Waldenstrom macroglobulinemia (WM) cells. We therefore investigated a role for HCK in mediating SYK activation. Overexpression of wild-type (WT) (HCKWT) or gatekeeper mutated (HCKThr333Met) HCK in MYD88Mut lymphoma cells triggered SYK activation. Conversely, HCK knockdown reduced p-SYK in MYD88Mut lymphoma cells. Coimmunoprecipitation experiments showed that HCK was complexed with p-SYK in MYD88Mut BCWM.1 and TMD8 cells, but not in MYD88 WT Ramos cells. Rescue experiments in MYD88Mut lymphoma cells expressing HCKThr333Met led to persistent HCK and SYK activation and resistance to the HCK inhibitor A419259. Treatment of primary MYD88Mut WM cells with A419259 reduced p-HCK and p-SYK expression. Taken together, our findings show that SYK is activated by HCK in MYD88Mut B-cell lymphomas cells, broaden the prosurvival signaling generated by aberrant HCK expression in response to MYD88Mut, and help define HCK as an important therapeutic target in MYD88Mut B-cell lymphomas.

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.

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.

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.

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.

Ibrutinib is associated with durable responses in patients with Waldenstrom macroglobulinaemia (WM). We hypothesized that response depth is predictive of progression-free survival (PFS) in WM patients treated with ibrutinib. Using landmark analyses, we evaluated response depth in two cohorts of WM patients treated with ibrutinib monotherapy. The learning cohort was composed of 93 participants from two clinical trials, and the validation cohort of 190 consecutive patients treated off clinical trial. Rates of partial response (PR) or better at six months in learning and validation cohorts were 64% and 71% respectively (P = 0·29). In the learning cohort, three-year PFS rates for patients who attained PR or better at six months versus not were 81% and 57% respectively (P = 0·009). In the validation cohort, three-year PFS rates for patients who attained PR or better at six months versus not were 83% and 54% respectively (P = 0·008). In multivariate analyses, attaining PR or better at six months was associated with superior PFS in the learning [hazard ratio (HR) 0·38; P = 0·01] and validation cohorts (HR 0·18; P = 0·004). Attaining PR at six months on ibrutinib emerges as an intermediate outcome of interest and should be validated as surrogate for PFS in clinical trials evaluating Bruton tyrosine kinase inhibitors in WM.

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.

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.

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.

Activating MYD88 mutations promote pro-survival signaling through BTK and HCK, both targets of ibrutinib. Despite high response rates, complete responses to ibrutinib are lacking, and other MYD88 triggered pro-survival pathways may contribute to primary drug resistance. B-cell receptor (BCR) signaling has been observed in lymphomas driven by mutated MYD88, even without activating the BCR pathway mutations. We identified activated SYK (p-SYK), a component of BCR in complex with MYD88 in MYD88-mutated WM and ABC DLBCL lymphoma cells. Confocal microscopy confirmed co-localization of MYD88 with SYK in MYD88-mutated cells. Knockdown of MYD88 or use of a MYD88 signaling inhibitor abrogated SYK activation, while expression of mutated but not wild-type MYD88 amplified p-SYK in MYD88-mutated and wild-type lymphoma cells. Knockdown of SYK or use of inhibitors targeting SYK blocked p-STAT3 and p-AKT signaling in MYD88-mutated cells. Cell viability analysis showed that combining ibrutinib and SYK inhibitors triggered synthetic killing of MYD88-mutated lymphoma cells. Our findings extend the spectrum of mutated MYD88 pro-survival signaling to include SYK directed BCR cross talk in MYD88-mutated lymphomas. Targeting SYK in combination with ibrutinib produces synthetic lethality, providing a framework for the clinical investigation of ibrutinib with SYK inhibitors in MYD88-mutated lymphomas.

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.

BACKGROUND: Multiple myeloma (MM) and Waldenström macroglobulinemia (WM) are associated with significant immunoparesis. Based on the ongoing COVID-19 pandemic, there is an urgent need to understand whether patients are able to mount a sufficient response to COVID-19 vaccines. METHODS: MM and WM patients are vaccinated with mRNA-1273 (Moderna), BNT162b2 mRNA (Pfizer/BioNTech), or JNJ-78436735 (Johnson & Johnson) in a prospective clinical trial. Primary endpoint is SARS-CoV-2 spike protein (S) antibody (Ab) detection 28 days after final vaccination. Secondary endpoints include functional serologic assessments and T-cell responses at 28 days, 6 months, 9 months, and 12 months following vaccination. S Abs were detected by Elecsys assay (Roche Diagnostics), with ³ 0.80 U/mL defined as positive and titers > 250 U/mL considered stronger correlates of neutralization. SARS-CoV-2 wildtype and variant S-specific Ab isotypes and FcγR binding profiles were quantified by custom Luminex assay. Antibody-dependent neutrophil and cellular phagocytosis (ADNP and ADCP) were assessed using flow cytometry. RESULTS: To date 141 patients have been enrolled, 137 (91 MM and 46 WM) of whom had an S Ab assessment. Median Ab titer was 178.0 (IQR, 16.10-1166.0) for MM and 3.92 (IQR, 0-278.9) for WM. S Ab response rate was 91% (83/91) in MM and 56% (27/46) in WM. However, responses achieving S Ab >250 U/mL were 47.3% (43/91) in MM and 26.1% (12/46) in WM. In patients ³75 years, responses >250 u/mL were 13.3% (2/15; p250 u/mL following mRNA-1273, BNT162b2, and JNJ-78436735 were 67.6% (23/34; p Among MM patients with progressive disease, S Ab response >250 u/mL occurred in 30% (6/20) as opposed to 55.6% (30/54) for VGPR+ (p Among WM patients, S Ab responses >250 U/mL occurred in 63.6% (7/11; p Functional Ab studies were performed on 14 MM patients, 14 WM, patients, and 14 healthy donors (HD) (Figure 1). All patients were assessed 28 days following their final vaccination and myeloma patients had an additional assessment 28 days following initial vaccination. MM and WM patients demonstrated less IGG1 and IGG3 S Ab production than HD. MM patients showed increased IgA and IgM S Ab production as well as increased FcgR2A binding following a second vaccine in contrast to HD. Both ADNP and ADCP were reduced in MM and WM patients. MM patients demonstrated improved ADCP in SARS-CoV-2 variants B.1.351, B.1.117, and P.1 versus wildtype (p CONCLUSIONS: We report the first known evidence of impaired functional humoral responses following COVID-19 vaccines in patients with MM and WM. Overall, WM patients showed more severe impairment of COVID-19 S Ab responses. Most previously untreated WM patients achieved S Ab responses, however the most significant reduction in S Ab responses were seen in WM patients who received rituximab within 12 months or active BTK inhibitors. For MM patients, being in disease remission associated with improved S Ab response. Among MM and WM patients, age ³75 years associated with significantly lower rates and vaccination with MRNA-1273 (Moderna) elicited significantly higher S Ab response rates than other vaccines. A defect in ADNP and more profound defect in ADCP suggests overall compromised opsinophagocytic activity among MM and WM patients. Data comparing first and second vaccine responses in MM patients, suggest less efficient class switching to IGG as well as incomplete maturation of their FcgR2A binding profiles but normal maturation of FcgR3A. Interestingly, ADCP was improved in several emerging SARS-CoV-2 variants. T-cell studies are pending and will be updated. Further understanding of the immunological response to COVID19 vaccination is needed to clarify patients risks, and necessity for booster or alternative protective measures against COVID-19. Figure 1 Figure 1. Disclosures Branagan: Adaptive Biotechnologies: Consultancy; BeiGene: Consultancy; CSL Behring: Consultancy; Karyopharm: Consultancy; Pharmacyclics: Consultancy; Sanofi Genzyme: Consultancy. Lei: AbbVie: Honoraria; Epizyme: Honoraria; Fresenius Kabi: Consultancy; Intervention Insights: Consultancy. Yee: Amgen: Consultancy; Bristol Myer Squibb: Consultancy; Adaptive: Consultancy; GSK: Consultancy; Janssen: Consultancy; Oncopeptides: Consultancy; Sanofi: Consultancy; Takeda: Consultancy; Karyopharm: Consultancy. O'Donnell: Adaptive: Consultancy; Bristol Myer Squibb: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Onocopeptide: Consultancy; Takeda: Consultancy. Castillo: Abbvie: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy; Roche: Consultancy; TG Therapeutics: Research Funding. Raje: Celgene, Amgen, Bluebird Bio, Janssen, Caribou, and BMS: Other. Treon: BeiGene: Consultancy, Research Funding; Eli Lily: Research Funding; Abbvie/Pharmacyclics: Consultancy, Research Funding. Mo: GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Epizyme: Consultancy; Eli Lilly: Consultancy; Janssen: Honoraria; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Nadeem: Takeda: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees. Richardson: Oncopeptides: Consultancy, Research Funding; Celgene/BMS: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Karyopharm: Consultancy, Research Funding; Protocol Intelligence: Consultancy; Janssen: Consultancy; Sanofi: Consultancy; Secura Bio: Consultancy; GlaxoSmithKline: Consultancy; Regeneron: Consultancy; AstraZeneca: Consultancy; AbbVie: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding. Juleg: Leyden Labs: Current Employment; Amsterdam: Current Employment; Netherlands: Current Employment.

MYD88 mutations are common in B-cell malignancies including Waldenstrom Macroglobulinemia (WM) and ABC subtype of Diffuse Large B-cell Lymphoma (ABC DLBCL). Mutated MYD88 activates BTK, and triggers downstream pro-survival signaling that includes NF-kB and ERK (Yang et al, Blood 2013; Blood 2016). ERK related signaling triggers inflammatory cytokine production including IL-6 and IL-10 (Chen et al, Blood 2016). Ibrutinib covalently binds to BTK Cys481 and inactivates BTK and downstream NF-kB and ERK signaling. Ibrutinib is approved for the treatment of WM and is associated with high overall response rates (>90%) and long term progression free survival in WM though intolerance to therapy, as well as resistance related to acquired BTK Cys481 mutations frequently leads to treatment discontinuation. We therefore investigated a novel, non-covalent BTK-inhibitor, pirtobrutinib that binds to BTK at non-Cys481 amino acids (G473-K483). Pirtobrutinib showed highly selective anti-proliferative activity against MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8 and HBL-1) versus MYD88 wild-type (OCI-Ly7, OCI-Ly19, Ramos, and RPMI-8226) cells, with marked apoptotic effect exhibited against primary MYD88 mutated WM cells at pharmacologically achievable levels (100-500 nM). Importantly, pirtobrutinib blocked BTK activity and overcame ibrutinib resistance in BCWM.1 WM and TMD-8 ABC DLBCL cells transduced to express both wild-type and mutated BTK (BTK Cys481Ser) with similar efficacy. The downstream signaling consequences of pirtobrutinib in vector only, wild-type and mutant BTK Cys481 expressing BCWM.1, MWCL-1, TMD-8 and HBL-1 cells was also examined. Treatment of vector only and wild-type BTK Cys481 expressing WM and ABC DLBCL cells with ibrutinib or pirtobrutinib abrogated both p-BTK and p-ERK signaling. In contrast, only pirtobrutinib blocked p-BTK and p-ERK signaling in mutant BTK Cys481 expressing WM and ABC DLBCL cells. In previous studies, we showed that inflammatory cytokine production that included IL-6 and IL-10 driven by ERK triggered ibrutinib resistance in wild-type BTK Cys481 MYD88 mutated lymphoma cells co-cultured with their mutated BTK expressing counterparts (Chen et al, BLOOD 2018). ERK-driven cytokine resistance to ibrutinib was postulated to explain how disease progression occurs in patients with modest variant expression of mutated BTK Cys481 (Woyach et al, JCO 2017; Xu et al, Blood 2017). Co-culture of BTK Cys481 mutated expressing TMD-8 cells with wild-type BTK expressing TMD-8 cells triggered resistance of the latter to ibrutinib. Treatment with pirtobrutinib blocked IL-6 and IL-10 production and overcame the protective effects conferred by BTK Cys481 mutated TMD-8 cells in these experiments. Lastly, oral administration of pirtobrutinib blocked p-BTK and p-ERK in BTK Cys481 mutated TMD-8 tumors xenografted in mice. Our findings therefore show that pirtobrutinib inhibits growth of MYD88 mutated lymphoma cells in a highly selective manner and can trigger apoptosis of primary WM patient BM lymphoplasmacytic cells at levels comparable to ibrutinib. Moreover, pirtobrutinib effectively blocked mutated BTK Cys481 driven BTK and ERK1/2 activation and produced similar cellular efficacy in both BTK wild-type and BTK Cys481 mutated cells. Pirtobrutinib also blocked the protective effect conferred to BTK wild-type cells through paracrine cytokines released by BTK Cys481Ser expressing cells. Lastly, pirtobrutinib blocked BTK and ERK1/2 activation in TMD8-BTK Cys481Ser xenografted mice. The findings support the development of pirtobrutinib in MYD88 driven lymphomas, including those resistant to ibrutinib on the bases of BTK Cys481 mutations. Disclosures Branagan: Adaptive Biotechnologies: Consultancy; BeiGene: Consultancy; CSL Behring: Consultancy; Karyopharm: Consultancy; Pharmacyclics: Consultancy; Sanofi Genzyme: Consultancy. Castillo: Abbvie: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy; Roche: Consultancy; TG Therapeutics: Research Funding. Yang: Blueprint Medicines Corporations: Current Employment, Current holder of individual stocks in a privately-held company. Treon: BeiGene: Consultancy, Research Funding; Eli Lily: Research Funding; Abbvie/Pharmacyclics: Consultancy, Research Funding.

Activating mutations in MYD88 (MYD88 Mut) are common in B-cell malignancies including Waldenstrom Macroglobulinemia (WM) and ABC subtype of diffuse B-cell lymphoma (ABC DLBCL). MYD88 is a component of the Toll-like receptor (TLR) pathway. We and others previously showed that MYD88 Mut triggers assembly of a "Myddosome" complex that leads to downstream pro-survival signaling that includes IRAK4/IRAK1 and BTK triggered NF-κB (Ngo et al, Nature 2011; Treon et al, NEJM 2012; Yang et al, Blood 2013) and HCK mediated BTK/NF-κB, PI3K/AKT, and MAPK/ERK signaling (Yang et al, Blood 2016; Liu et al Blood Adv. 2020). The activation of the B-cell receptor (BCR) signaling component SYK has also been observed in MYD88 Mut WM (Argyropoulos et al, Leukemia 2016). In ABC DLBCL, chronic active BCR signaling underlies SYK activation that is triggered by the SRC family member LYN (Davis et al, Nature 2010). These observations led us to explore potential drivers of BCR/SYK activation in WM. We previously reported that MYD88 Mut triggered activation of SYK in WM and ABC DLBCL cells (Munshi et al, BCJ 2020). Herein, we investigated if HCK, a SRC family member that is transcriptionally upregulated and activated by MYD88 Mut could trigger the BCR pathway through SYK activation. Since LYN is an integral part of BCR signaling, we first examined its expression and activation state in MYD88 Mut WM and ABC DLBCL cells. While MYD88 Mut TMD8, HBL-1 and OCI-Ly3 ABC DLBCL cells showed strong expression of p-LYN, such expression was absent or low in MYD88 Mut BCWM.1 and MWCL-1 cells, as well as CD19-selected bone marrow derived primary lymphoplasmacytic cells (LPCs) from WM patients. In view of the above findings, we next interrogated a direct role for HCK in mediating SYK activation. We over-expressed wild-type HCK (HCK WT) or gatekeeper mutated HCK (HCK T333M) in MYD88 Mut BCWM.1 and MWCL-1 WM cell lines, and TMD8 ABC DLBCL cells. In all these cell lines, over-expression of HCK WT or HCK T333M triggered a robust increase in phosphorylation of SYK Y525/Y526 in comparison to vector only transduced cells. Moreover, using an inducible vector system, knockdown of HCK showed a marked reduction in phosphorylation of SYK Y525/Y526 in MYD88 Mut BCWM.1 WM and TMD8 ABC DLBCL cells. We next sought to clarify if HCK and activated SYK were present in the same signaling complex. We performed co-immunoprecipitation experiments using an HCK antibody in MYD88 Mut BCWM.1, TMD8 and wild-type MYD88 (MYD88 WT) Ramos cells. The HCK antibody effectively pulled down p-SYK in MYD88 Mut BCWM.1 and TMD8 cells, but not in MYD88 WT Ramos cells. To confirm whether SYK activation was a result of HCK kinase activity, we next performed rescue experiments with the HCK inhibitors A419259 and KIN-8194 in MYD88 Mut BCWM.1 and MWCL-1 WM and TMD8 ABC DLBCL cells expressing either HCK WT or the HCK T333M protein that abrogated the activity of these inhibitors against HCK. Expression of the HCK T333M protein produced marked resistance to A419259 as well as KIN-8194 versus vector or HCK WT transduced BCWM.1 and MWCL-1 cells. By PhosFlow analysis, we observed that expression of HCK T333M but not HCK WT led to persistent activation of HCK and SYK in the presence of A419259 or KIN-8194 in BCWM.1 and MWCL-1 WM cells, and TMD8 ABC DLBCL cells. Consistent with these observations, treatment of primary MYD88 mutated WM LPCs cells with either A419259 or KIN-8194 also showed marked reduction in both p-HCK and p-SYK expression by PhosFlow analysis. Taken together, our findings show that SYK is activated by HCK in MYD88 Mut B-cell lymphomas cells; broaden the pro-survival signaling generated by aberrant HCK expression in response to MYD88 Mut; and help further establish HCK as an important therapeutic target in MYD88 Mut B-cell lymphomas. Disclosures Palomba: Juno: Patents & Royalties; Rheos: Honoraria; Seres: Honoraria, Other: Stock, Patents & Royalties, Research Funding; Notch: Honoraria, Other: Stock; Kite: Consultancy; Novartis: Consultancy; BeiGene: Consultancy; Priothera: Honoraria; Nektar: Honoraria; PCYC: Consultancy; Wolters Kluwer: Patents & Royalties; WindMIL: Honoraria; Magenta: Honoraria; Pluto: Honoraria; Lygenesis: Honoraria; Ceramedix: Honoraria. Castillo: Abbvie: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy; Roche: Consultancy; TG Therapeutics: Research Funding. Gray: Syros, C4, Allorion, Jengu, B2S, Inception, EoCys, Larkspur (board member) and Soltego (board member: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Takeda, Astellas, Taiho, Jansen, Kinogen, Arbella, Deerfield and Sanofi: Research Funding. Munshi: Bristol-Myers Squibb: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Takeda: Consultancy; Karyopharm: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Legend: Consultancy; Pfizer: Consultancy. Anderson: Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Yang: Blueprint Medicines Corporations: Current Employment, Current holder of individual stocks in a privately-held company. Treon: BeiGene: Consultancy, Research Funding; Eli Lily: Research Funding; Abbvie/Pharmacyclics: Consultancy, Research Funding.

MYD88 mutations (MYD88 MUT) are present in 95-97% of patients with Waldenström's Macroglobulinemia (WM). Transgenic mouse studies show that MYD88 MUT alone is insufficient to drive oncogenesis. We previously reported the occurrence of non-overlapping somatic mutations in either CXCR4 (CXCR4 MUT) or deletions in 6q (del6q) in MYD88 mutated (MYD88 MUT) WM patients, suggesting either event may serve as a secondary driver of WM oncogenesis. Intersecting the lists of genes differentially modulated by either CXCR4 MUT or del6q revealed WNK2 as a top hit (Guerrera ML et al, Haematologica 2018). WNK2 is a serine/threonine protein kinase that negatively regulates ERK1/2 activation and has known tumor suppressor function in several solid tumors, wherein its expression is primarily silenced by aberrant promoter methylation. ERK1/2 pathway plays an important pro-survival role, its activation is accompanied by the release of inflammatory cytokines and can mediate ibrutinib resistance in WM (Chen et al, Blood 2018). We therefore sought to investigate the expression of WNK2, and clarify the mechanisms underlying its aberrant expression by genomic subtypes in WM. To study the regulation of WNK2 gene expression, we analyzed our previously published RNA-Seq data (Hunter ZH et al, Blood 2016) and validated findings in 47 WM patients by RT-qPCR. Compared to healthy donor (HD) B and plasma cells (PC), we observed marked upregulation of WNK2 expression in bone marrow (BM) CD19-selected tumor cells from MYD88 MUTCXCR4 WT patients (p To assess the impact of alternative splice variants on overall gene expression, we then studied the relative composition of the 16 documented isoforms (Figure 1). RNA-Seq showed a prevalence of protein-coding transcripts, particularly those coding for isoforms missing the ERK regulatory kinase domain, in MYD88 MUT vs MYD88 WT WM. By RT-PCR, we validated the preferential use of the protein-coding WNK2-208, WNK2-203 and WNK2-205 transcripts in 32 of 47 WM samples with detectable total WNK2 levels. Of those, WNK2-208 and WNK2-203 lack the region coding for the functional kinase domain. Analysis of known post-translational modification (PTM) sites annotated for the canonical isoform WNK2-201 in the 3 isoforms of interest demonstrated that C-terminal PTM sites are highly conserved across all isoforms even in the absence of the kinase domain, alluding to novel protein regulatory sites yet to be characterized. Analysis of CpG methylation using enhanced reduced representation bisulfite sequencing from 62 WM patients identified 3 clusters of differentially methylated CpG islands (CpGs) between MYD88 MUTCXCR4 WT and MYD88 MUTCXCR4 MUT patients. Three CpGs in the WNK2 promoter, four in intron 1 and one in intron 11 constituted clusters 1, 2 and 3, respectively (Figure 1). Compared to healthy donor memory B-cell and PC, we observed aberrant hypermethylation in cluster 1 in MYD88 MUTCXCR4 MUT and hypomethylation at a putative enhancer in cluster 2 in MYD88 MUTCXCR4 WT patients, while del6q cases showed a mixed methylation pattern. A higher degree of methylation, particularly in cluster 2, significantly correlated with reduction in total and transcript-specific WNK2 mRNA levels that affected expression of transcripts encoding for kinase domain-containing isoforms. Furthermore, we validated the differential methylation status of 3 of 4 methylated CpGs in cluster 2 in 10 patients, including MYD88 MUTCXCR4 WT, MYD88 MUTCXCR4 MUT and MYD88 MUTdel6q, and 3 MYD88 MUT(BCWM.1, TMD-8) and MYD88 WT (Ramos) cell lines by means of bisulfite PCR followed by Sanger sequencing. Taken together, our findings reveal that the ERK1/2 regulator WNK2 shows differential expression and isoform usage in distinct genomic subsets of MYD88 MUT WM; is primarily driven by aberrant methylation; and may serve as a key driver of disease progression in WM. Figure 1 Figure 1. Disclosures Yang: Blueprint Medicines Corporations: Current Employment, Current holder of individual stocks in a privately-held company. Castillo: Abbvie: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy; Roche: Consultancy; TG Therapeutics: Research Funding. Treon: BeiGene: Consultancy, Research Funding; Eli Lily: Research Funding; Abbvie/Pharmacyclics: Consultancy, Research Funding.

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

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

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.

Waldenstrom’s Macroglobulinemia (WM) is a lymphoplasmacytic lymphoma characterized by bone marrow (BM) infiltration of immunoglobulin M (IgM)-secreting lymphoplasmacytic cells.[1][1] Activating mutations in MYD88 are present in 93-97% of WM and 50-70% of IgM monoclonal gammopathy of undetermined

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.

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.

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.

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.

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.

High-dose chemotherapy with autologous stem cell transplantation (ASCT) has been shown effective in the control of relapsed/refractory follicular lymphoma. We evaluate the long-term outcome of patients with relapsed or refractory follicular lymphoma treated with ASCT with in vivo purged progenitors cells. We report the long-term results of a prospective multicenter phase 2 trial on 124 relapsed/refractory follicular lymphoma patients treated with a program of anthracycline-based debulking chemotherapy, immunochemotherapy, mobilization of in vivo purged PBSC followed by ASCT. Median age was 52 years; 14% of patients had grade 3A histology. Debulking chemotherapy produced CR in 16% and PR in 71%, while 13% of patients progressed. After rituximab, cyclophosphamide, vincristine, prednisone (R-COP), CR was obtained in 60% and PR in 35%; 118 patients successfully mobilized PBSC and 117 proceeded to ASCT. The harvest in all the 32 molecularly informative patients was bcl-2 negative. TRM was 0%. The 5-year PFS was 54% and the 5-year OS was 83%. After a median f-up of 6.7 years (range 1.5-13.6), 54% are still in CR. These data show that prolonged PFS is achievable in relapsed/refractory patients with high dose autologous transplantation of in vivo purged progenitor cells.

Acquired ibrutinib resistance due to BTK

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.