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Acute lymphoblastic leukemia (ALL) harboring the IgH-CRLF2 rearrangement (IgH-CRLF2-r) exhibits poor clinical outcomes and is the most common subtype of Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL). While multiple chemotherapeutic regimens, including ruxolitinib monotherapy and/or its combination with chemotherapy, are being tested, their efficacy is reportedly limited. To identify molecules/pathways relevant for IgH-CRLF2-r ALL pathogenesis, we performed genome-wide CRISPR-Cas9 dropout screens in the presence or absence of ruxolitinib using 2 IgH-CRLF2-r ALL lines that differ in RAS mutational status. To do so, we employed a baboon envelope pseudotyped lentiviral vector system, which enabled, for the first time, highly efficient transduction of human B cells. While single-guide RNAs (sgRNAs) targeting CRLF2, IL7RA, or JAK1/2 significantly affected cell fitness in both lines, those targeting STAT5A, STAT5B, or STAT3 did not, suggesting that STAT signaling is largely dispensable for IgH-CRLF2-r ALL cell survival. We show that regulators of RAS signaling are critical for cell fitness and ruxolitinib sensitivity and that CRKL depletion enhances ruxolitinib sensitivity in RAS wild-type (WT) cells. Gilteritinib, a pan-tyrosine kinase inhibitor that blocks CRKL phosphorylation, effectively killed RAS WT IgH-CRLF2-r ALL cells in vitro and in vivo, either alone or combined with ruxolitinib. We further show that combining gilteritinib with trametinib, a MEK1/2 inhibitor, is an effective means to target IgH-CRLF2-r ALL cells regardless of RAS mutational status. Our study delineates molecules/pathways relevant for CRLF2-r ALL pathogenesis and could suggest rationally designed combination therapies appropriate for disease subtypes.

The basis for acquired resistance to JAK inhibition in patients with JAK2-driven hematologic malignancies is not well understood. We report a patient with a myeloproliferative neoplasm (MPN) with a BCR activator of RhoGEF and GTPase (BCR)–JAK2 fusion with initial hematologic response to ruxolitinib who rapidly developed B-lymphoid blast transformation. We analyzed pre-ruxolitinib and blast transformation samples using genome sequencing, DNA mate-pair sequencing (MPseq), RNA sequencing (RNA-seq), and chromosomal microarray to characterize possible mechanisms of resistance. No resistance mutations in the BCR-JAK2 fusion gene or transcript were identified, and fusion transcript expression levels remained stable. However, at the time of blast transformation, MPseq detected a new IKZF1 copy-number loss, which is predicted to result in loss of normal IKZF1 protein translation. RNA-seq revealed significant upregulation of genes negatively regulated by IKZF1, including IL7R and CRLF2. Disease progression was also characterized by adaptation to an activated B-cell receptor (BCR)–like signaling phenotype, with marked upregulation of genes such as CD79A, CD79B, IGLL1, VPREB1, BLNK, ZAP70, RAG1, and RAG2. In summary, IKZF1 deletion and a switch from cytokine dependence to activated BCR-like signaling phenotype represent putative mechanisms of ruxolitinib resistance in this case, recapitulating preclinical data on resistance to JAK inhibition in CRLF2-rearranged Philadelphia chromosome-like acute lymphoblastic leukemia.

Hemophagocytic lymphohistiocytosis (HLH) is an inflammatory disorder in which numerous cytokines are elevated, though interferon-γ (IFN-γ) is central to disease pathogenesis and a key therapeutic target. Experimental and early clinical reports have shown that ruxolitinib, a small molecule inhibitor of Janus kinases (JAKs), which are essential for cytokine signaling, may be therapeutic in HLH. In contrast, we found that intermittently administered ruxolitinib at various dose levels failed to prevent HLH development or treat established murine HLH. High doses of ruxolitinib blocked IFN-γ signaling only transiently after administration, consistent with human pharmacokinetics, and only continuously administered drug could prevent HLH development or treat established HLH. Continuously administered ruxolitinib was therapeutic in only a narrow dose range and intermittently dosed ruxolitinib worsened survival and decreased bone marrow cellularity of animals concurrently treated with anti-IFN-γ antibody, indicating a narrow therapeutic window and potential toxicity. Because JAK2 is essential for hematopoietic cytokine signaling, we also tested a JAK1-selective inhibitor and observed therapeutic benefit without apparent toxicity, though it did not improve survival when combined with anti-IFN-γ. We conclude that continuous blockade of IFN-γ signaling is necessary for optimal control of HLH and that JAK2 inhibition may be toxic in this disorder.

Marginal zone lymphoma (MZL) is challenging to treat, with many patients relapsing following initial treatment. We report the long-term efficacy and safety of copanlisib, a pan-class I phosphoinositide 3-kinase (PI3K) inhibitor, in the subset of 23 patients with relapsed/refractory MZL treated in the phase 2 CHRONOS-1 study (#NCT01660451, Part B; www.clinicaltrials.gov). Patients had a median of 3 prior lines of therapy, including rituximab and alkylating agents, and received IV copanlisib 60 mg on days 1, 8, and 15 of 28-day cycles for a median of 23 weeks. The objective response rate was 78.3% (18/23; 3 complete responses and 15 partial responses). The median duration of response was 17.4 months (median follow-up, 9.4 months), and median time to response was 2.1 months. Median progression-free survival was 24.1 months (median follow-up, 10.3 months), and median overall survival was not reached (median follow-up, 28.4 months). The most common all-grade treatment-emergent adverse events (TEAEs) included fatigue (52.2%, 12/23), diarrhea, and transient, infusion-related hyperglycemia (each 47.8%, 11/23). Nineteen patients (82.6%) had grade 3/4 TEAEs, most commonly transient, infusion-related hyperglycemia and hypertension (each 39.1%, 9/23). TEAEs led to dose reduction or dose interruptions /delays in 9 patients (39.1%) and 18 patients (78.3%), respectively. Patients with activated PI3K/B-cell antigen receptor signaling had improved response rates. Overall, copanlisib demonstrated strong efficacy, with a short time to objective response, improved objective response rate with longer treatment duration, durable responses, and manageable safety, in line with previous reports. These data provide rationale for long-term treatment with copanlisib in patients with relapsed/refractory MZL.

Zanubrutinib (BGB-3111) is a next-generation Bruton tyrosine kinase inhibitor designed to be more selective with fewer off-target effects. We conducted a phase 1 study to assess the safety of its combination with obinutuzumab and evaluate early efficacy in 81 patients with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) or relapsed/refractory (R/R) follicular lymphoma (FL). In this phase 1b study, zanubrutinib was tolerable at 160 mg twice daily or 320 mg once daily combined with IV obinutuzumab in patients with CLL/SLL (n = 45) and FL (n = 36). Common adverse events (AEs) included upper respiratory tract infection (51%; n = 23), neutropenia (44%; n = 20), contusion (33%; n = 15), cough, diarrhea, or fatigue (27%; n = 12 each), and pyrexia (22%; n = 10) in CLL/SLL patients and upper respiratory tract infection (39%; n = 14), contusion (28%; n = 10), fatigue (25%; n = 9), and cough (22%; n = 8) in FL patients. Neutropenia was the most common grade 3/4 AE (CLL/SLL, 31% [n = 14]; FL, 14% [n = 5]). Five patients required temporary dose reductions, and 5 discontinued the study drug because of AEs. Overall response rate (ORR) was 100% (n = 20) in treatment-naïve CLL patients and 92% (n = 23) in R/R CLL patients. ORR in 36 R/R FL patients was 72% (n = 26), with 14 complete and 12 partial responses. Median follow-up was 29 months (range, 8-37) for CLL patients and 20 months (range, 2-37) for FL patients. Zanubrutinib and obinutuzumab combination therapy was generally well tolerated. This trial was registered at www.clinicaltrials.gov as #NCT02569476.

Ruxolitinib is a recommended second-line treatment for the prevention of thrombosis in patients with polycythemia vera who become resistant or intolerant to hydroxyurea; however, evidence regarding its efficacy in terms of thrombosis reduction is uncertain. We searched Medline, Embase, and archives of abstracts from the European Hematology Association and the American Society of Hematology annual congresses from 2014 onward for randomized controlled trials comparing the treatment vs best available therapy (BAT). Our search retrieved 80 records; after screening of abstracts and full text, the total was reduced to 16. Evidence came from 4 randomized controlled trials, including 663 patients (1057 patients per year). We estimated a thrombosis risk ratio of 0.56 for ruxolitinib BAT, corresponding to an incidence of 3.09% and 5.51% patients per year, respectively. The number of thrombotic events reported with ruxolitinib was consistently lower than that with BAT in our sample, but, globally, the difference did not reach significance (P = .098). Hard evidence in favor of ruxolitinib is lacking; a clinical trial on selected patients at high risk of thrombosis would be warranted, but its feasibility is questionable.

Aberrant JAK2 tyrosine kinase signaling drives the development of Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. However, JAK2 kinase inhibitors have failed to significantly reduce allele burden in MPN patients, underscoring the need for improved therapeutic strategies. Members of the PIM family of serine/threonine kinases promote cellular proliferation by regulating a variety of cellular processes, including protein synthesis and the balance of signaling that regulates apoptosis. Overexpression of PIM family members is oncogenic, exemplified by their ability to induce lymphomas in collaboration with c-Myc. Thus, PIM kinases are potential therapeutic targets for several malignancies such as solid tumors and blood cancers. We and others have shown that PIM inhibitors augment the efficacy of JAK2 inhibitors by using in vitro models of MPNs. Here we report that the recently developed pan-PIM inhibitor INCB053914 augments the efficacy of the US Food and Drug Administration-approved JAK1/2 inhibitor ruxolitinib in both in vitro and in vivo MPN models. INCB053914 synergizes with ruxolitinib to inhibit cell growth in JAK2-driven MPN models and induce apoptosis. Significantly, low nanomolar INCB053914 enhances the efficacy of ruxolitinib to inhibit the neoplastic growth of primary MPN patient cells, and INCB053914 antagonizes ruxolitinib persistent myeloproliferation in vivo. These findings support the notion that INCB053914, which is currently in clinical trials in patients with advanced hematologic malignancies, in combination with ruxolitinib may be effective in MPN patients, and they support the clinical testing of this combination in MPN patients.

Ibrutinib, a once-daily oral inhibitor of Bruton tyrosine kinase, has greatly improved outcomes for patients with chronic lymphocytic leukemia (CLL). The phase 3 RESONATE trial, which compared single-agent ibrutinib to ofatumumab in high-risk, relapsed patients with CLL, provided support for approval of ibrutinib in the United States and Europe. We describe long-term follow-up of patients treated in RESONATE, where continued superiority of progression-free survival (PFS) (hazard ratio [HR], 0.133; 95% confidence interval [CI], 0.099-0.178) was observed. Overall survival benefit continues (HR, 0.591; 95% CI, 0.378-0.926), although with decreased magnitude relative to that seen before crossover to ibrutinib was implemented for patients on ofatumumab (HR, 0.426; 95% CI, 0.220-0.823). Notably, overall response to ibrutinib increased over time, with 91% of patients attaining a response. The PFS benefit with ibrutinib was independent of baseline risk factors, although patients with ≥2 prior therapies had shorter PFS than those with

Although second-generation tyrosine kinase inhibitors (TKIs) show superiority in achieving deep molecular responses in chronic myeloid leukemia in chronic phase (CML-CP) compared with imatinib, the differing adverse effect (AE) profiles need consideration when deciding the best drug for individual patients. Long-term data from randomized trials of nilotinib demonstrate an increased risk of vascular AEs (VAEs) compared with other TKIs, although the natural history of these events in response to dose modifications or cessation has not been fully characterized. We retrospectively reviewed the incidence of nilotinib-associated AEs in 220 patients with CML-CP at 17 Australian institutions. Overall, AEs of any grade were reported in 95 patients (43%) and prompted nilotinib cessation in 46 (21%). VAEs occurred in 26 patients (12%), with an incidence of 4.1 events per 100 patient-years. Multivariate analysis identified age (P = .022) and dyslipidemia (P = .007) as independent variables for their development. There was 1 fatal first VAE, whereas the remaining patients either continued nilotinib (14 patients) or stopped it immediately (11 patients). Recurrent VAEs were associated with ongoing therapy in 7 of 14 who continued (with 2 fatal VAEs) vs 1 of 11 who discontinued (P = .04). Nineteen of the 23 evaluable patients surviving a VAE ultimately stopped nilotinib, of whom 14 received an alternative TKI. Dose reduction or cessation because of VAEs did not adversely affect maintenance of major molecular response. These findings demonstrate that in contrast to other AEs, VAEs are ideally managed with nilotinib cessation because of the increased risk of additional events with its ongoing use.

Cardiovascular or arteriothrombotic adverse events (CV- or AT-AEs) are reported in chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors (TKIs). The incidence and characteristics across different TKI have not been systematically analyzed. We analyzed 531 patients treated with frontline TKIs in different prospective trials: imatinib 400 mg (n = 71) and 800 mg (n = 203), nilotinib (n = 108), dasatinib (n = 106), and ponatinib (n = 43). Characteristics and incidence of new-onset CV-AEs and AT-AEs were analyzed. Poisson regression models assessed factors associated with AE incidence. Median follow-up was 94 months (range, 2-195). Overall, 237 patients (45%) developed CV-AEs and 46 (9%) developed AT-AEs. Hypertension was the most common AE seen in 175 patients (33%; grade 3/4 in 17%). CV-AE and AT-AE incidence ratios (IRs) with 95% confidence intervals (CIs) were 8.6 (7.6-9.8) and 1.7 (1.2-2.2) per 100 person-years. Among the TKIs, ponatinib showed the highest IR (95% CI) for CV-AEs and AT-AEs at 40.7 (27.9-59.4) and 9.0 (4.1-20.1). In multivariate analysis, ponatinib therapy was associated with increased incidence rate ratio (IRR) for CV-AEs (4.62; 95% CI, 2.7-7.7; P < .0001) and AT-AEs (6.38; 95% CI, 1.8-21.8; P < .0001) compared with imatinib 400. In summary, there is an increased risk of CV-AEs (except hypertension) and AT-AEs in CML patients treated with newer TKIs, particularly with ponatinib. Patients on TKIs must be informed and closely monitored for vascular AEs. These studies were registered at www.clinicaltrials.gov as #NCT00048672, #NCT00038649, #NCT00050531, #NCT00254423, #NCT00129740, and #NCT01570868.

Ibrutinib, an oral covalent inhibitor of Bruton’s tyrosine kinase, is an effective therapy for patients with chronic lymphocytic leukemia (CLL). To determine whether rituximab provides added benefit to ibrutinib, we conducted a randomized single-center trial of ibrutinib vs ibrutinib plus rituximab. Patients with CLL requiring therapy were randomized to receive 28-day cycles of once-daily ibrutinib 420 mg, either as a single agent (n = 104), or together with rituximab (375 mg/m2; n = 104), given weekly during cycle 1, then once per cycle until cycle 6. The primary end point was progression-free survival (PFS) in the intention-to-treat population. We enrolled 208 patients with CLL, 181 with relapsed CLL and 27 treatment-naive patients with high-risk disease (17p deletion or TP53 mutation). After a median follow-up of 36 months, the Kaplan-Meier estimates of PFS were 86% (95% confidence interval [CI], 76.6-91.9) for patients receiving ibrutinib, and 86.9% (95% CI, 77.3-92.6) for patients receiving ibrutinib plus rituximab. Similarly, response rates were the same in both arms (overall response rate, 92%). However, time to normalization of peripheral blood lymphocyte counts and time to complete remission were shorter, and residual disease levels in the bone marrow were lower, in patients receiving ibrutinib plus rituximab. We conclude that the addition of rituximab to ibrutinib in relapsed and treatment-naive high-risk patients with CLL failed to show improvement in PFS. However, patients treated with ibrutinib plus rituximab reached their remissions faster and achieved significantly lower residual disease levels. Given these results, ibrutinib as single-agent therapy remains current standard-of-care treatment in CLL. This trial was registered at www.clinicaltrials.gov as #NCT02007044.

Targeted therapies including the engineered afucosylated anti-CD20 monoclonal antibody obinutuzumab, Bruton’s tyrosine kinase inhibitor ibrutinib, and B-cell lymphoma protein 2 inhibitor venetoclax have demonstrated significant clinical activity in chronic lymphocytic leukemia (CLL) and, based on their complementary mechanisms, are ideal for combination. However, combining venetoclax with other active agents raises safety concerns, as it may increase the risk for tumor lysis syndrome. To minimize this risk, we designed and implemented a fixed-duration regimen using sequentially administered obinutuzumab followed by ibrutinib (cycle 2) and venetoclax (cycle 3), for a total of fourteen 28-day cycles. This phase 1b study included 12 patients with relapsed or refractory CLL. We tested 3 dose levels of venetoclax and identified the doses of all 3 agents approved by the US Food and Drug Administration for use in the combination. Adverse events were consistent with known toxicities of the individual agents, with hematologic adverse events being most frequent. No clinically significant tumor lysis syndrome occurred. The overall response rate was 92% (95% confidence interval, 62%-100%), with 42% (5/12) achieving a complete remission or complete remission with incomplete marrow recovery. There were 6 patients with no detectable CLL in both the blood and bone marrow at the end of treatment. We found this regimen to be safe and tolerable in CLL, and capable of inducing deep responses, justifying future study in our ongoing phase 2 cohorts of relapsed or refractory and treatment-naive patients, as well as larger phase 3 trials currently in planning. This trial was registered at www.clinicaltrials.gov as #NCT02427451.

Imatinib is the mainstay of treatment of patients with chronic myeloid leukemia (CML) in Tanzania. Monitoring molecular response to therapy by real-time polymerase chain reaction at defined milestones is necessary for early detection of treatment failure. However, this assay is not routinely performed in Tanzania; therefore, the depth of molecular response among patients with CML is not known. A total of 158 patients with previously diagnosed CML who received imatinib treatment were recruited from January 2019 and followed up through October 2020 at Ocean Road Cancer Institute. Information was obtained at the time of diagnosis and follow-up. Blood samples were collected in EDTA tubes to measure the BCR/ABL ratio on the Gene Xpert system for molecular response determination. The median age of the 158 adult patients was 45 years (range, 18-86). By reference to established treatment milestones, only 37 (23.4%) achieved optimal molecular response. Signs of advanced-stage disease, in particular the need for red cell transfusions before diagnosis (adjusted odds ratio [AOR], 3.4; 95% CI, 1.32-9.17) and cytopenias (AOR, 2.26; 95% CI, 1.03-4.96) necessitating drug interruptions were statistically validated predictors of treatment failure on multivariate, multinomial logistic regression. Patient survival at the 22-month follow-up was lowest, with 78.6% (95% CI, 69.4-85.4) in the failure-to-respond category and highest in patients achieving optimal response 97.0% (95% CI, 80.9-99.6). In summary, the majority of patients with CML treated with imatinib in Tanzania do not obtain deep molecular response. This outcome can be attributed to late diagnosis, the development of cytopenias requiring multiple drug interruptions, and poor adherence to treatment.

PAC203 is a randomized dose-finding study of pacritinib, an oral JAK2/IRAK1 inhibitor, in patients with advanced myelofibrosis who are intolerant of or resistant to ruxolitinib. Patients were randomized 1:1:1 to pacritinib 100 mg once per day, 100 mg twice per day, or 200 mg twice per day. Enhanced eligibility criteria, monitoring, and dose modifications were implemented to mitigate risk of cardiac and hemorrhagic events. Efficacy was based on ≥35% spleen volume response (SVR) and ≥50% reduction in the 7-component total symptom score (TSS) through week 24. Of 161 patients, 73% were intolerant of and 76% had become resistant to ruxolitinib; 50% met criteria for both. Severe thrombocytopenia (platelet count

Myeloproliferative neoplasms (MPN) that have evolved into accelerated or blast phase disease (MPN-AP/BP) have poor outcomes with limited treatment options and therefore represent an urgent unmet need. We have previously demonstrated in a multicenter, phase 1 trial conducted through the Myeloproliferative Neoplasms Research Consortium that the combination of ruxolitinib and decitabine is safe and tolerable and is associated with a favorable overall survival (OS). In this phase 2 trial, 25 patients with MPN-AP/BP were treated at the recommended phase 2 dose of ruxolitinib 25 mg twice daily for the induction cycle followed by 10 mg twice daily for subsequent cycles in combination with decitabine 20 mg/m2 for 5 consecutive days in a 28-day cycle. Nineteen patients died during the study follow-up. The median OS for all patients on study was 9.5 months (95% confidence interval, 4.3-12.0). Overall response rate (complete remission + incomplete platelet recovery + partial remission) was 11/25 (44%) and response was not associated with improved survival. We conclude that the combination of decitabine and ruxolitinib was well tolerated, demonstrated favorable OS, and represents a therapeutic option for this high-risk patient population. This trial was registered at www.clinicaltrials.gov as #NCT02076191.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a distinct subset of cutaneous lymphomas in which neoplastic cytotoxic α/β CD8+ T cells infiltrate subcutaneous adipose tissue.1-3 SPTCL may affect both children and adults.4 Systemic symptoms such as fever, asthenia, and weight loss, associated with cytopenias and liver dysfunction, are common. In addition, hemophagocytic lymphohistiocytosis (HLH) is observed in 20% of cases,3 which is associated with poor prognosis and may be the leading cause of death.3 There is no standardized therapy for SPTCL alone or in association with HLH. Chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisolone is frequently used, with an overall remission rate of 50%.1 Immunosuppressive regimens, particularly cyclosporine A (CsA), may be also effective.5 In some severe cases, stem cell transplantation has been attempted.1 Recently, germline mutations causing loss of function of T-cell immunoglobulin mucin 3 (TIM-3) were identified in 60% to 85% of SPTCL patients.6,7 In these patients, TIM-3 deficiency was shown to promote T-lymphocyte and -macrophage activation and the production of proinflammatory cytokines, challenging the malignant nature of skin T-lymphocyte infiltration.6 Ruxolitinib is a selective JAK1/JAK2 inhibitor licensed for treatment of myelofibrosis and polycythemia vera in adults.8,9 Studies in animal models of HLH support the efficacy of this drug to prevent and treat HLH in these models.10,11 Anecdotal experiences of successful use of ruxolitinib to control refractory primary HLH or secondary HLH are also reported in humans.12-19 JAK1/JAK2 inhibitors are also increasingly used in inflammatory diseases. We herein report the use and efficacy of ruxolitinib in a patient with recurrence of SPTCL and HLH and in whom TIM-3 deficiency was recently identified.6

Continuous treatment vs fixed duration of monotherapies and combinations of targeted agents are treatment options in relapsed chronic lymphocytic leukemia. The optimal choice of relapse treatment is dependent on the prior frontline therapy, duration of remission after frontline, genetic markers, and patients’ condition, including age and comorbidities. Combination therapies may result in deep responses with undetectable minimal residual disease (uMRD). Although uMRD is an excellent predictive marker for disease progression, it is rarely used in clinical practice and needs additional evaluation in clinical trials before discontinuation of therapy should be guided according to uMRD.

Attempts to improve upon the activity of ibrutinib in chronic lymphocytic leukemia (CLL) include the addition of targeted therapies. The combination of lenalidomide and rituximab demonstrated an overall response rate (ORR) of 66% with a complete response (CR) of 12% in the relapsed/refractory setting. Based on these data, we conducted a phase 1 study of rituximab (R), lenalidomide (L), and ibrutinib (I) in relapsed/refractory CLL. Patients received R 375 mg/m2 cycles 1 to 6 day 1, L on cycles 1 to 12 days 1 to 21, and I until disease progression. Dose escalation used a standard 3+3 design from a dose level (DL) of L 5 mg (DL1) and increasing to 15 mg (DL3) for a total of 3 dose levels. Twelve patients were enrolled; there were 2 dose-limiting toxicities of grade 4 neutropenia at DL3; thus, DL2 was the recommended phase 2 dose. A high incidence of sustained grade 4 neutropenia occurred at all dose levels, prompting study withdrawal in 5 patients, despite growth factor support. The ORR was 67%; ORR at the RP2D was 100% (1 CR). The 12-month progression-free survival at the RP2D was 83%. Preliminary efficacy data with the triplet did not appear superior to prior reports of the rituximab-lenalidomide doublet or single-agent ibrutinib. Given these findings and the sustained neutropenia, this regimen was not pursued. The study was registered at www.clinicaltrials.gov as #NCT02200848.

Pevonedistat (TAK-924/MLN4924) is a novel inhibitor of NEDD8-activating enzyme (NAE) with single-agent activity in relapsed/refractory acute myeloid leukemia (AML). We performed a phase 1b study of pevonedistat (PEV) with azacitidine (AZA) based on synergistic activity seen preclinically. Primary objectives included safety and tolerability, and secondary objectives included pharmacokinetics (PK) and disease response. Patients ≥60 years with treatment-naive AML (unfit for standard induction therapy) received PEV 20 or 30 mg/m2 IV on days 1, 3, and 5 combined with fixed-dose AZA (75 mg/m2 IV/subcutaneously) on days 1 to 5, 8, and 9, every 28 days. The most common treatment-emergent adverse events were constipation (48%), nausea (42%), fatigue (42%), and anemia (39%). In total, 11 deaths were observed and considered unrelated to study therapy by the investigators. Transient elevations in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were dose limiting. The recommended phase 2 dose (RP2D) of PEV in this combination is 20 mg/m2 PEV PK was not altered by the addition of AZA. Overall response rate (ORR) based on an intent-to-treat analysis was 50% (20 complete remissions [CRs], 5 complete remission with incomplete peripheral count recovery, 7 partial remissions [PRs]), with an 8.3-month median duration of remission. In patients receiving ≥6 cycles of therapy (n = 23, 44%), ORR was 83%. In patients with TP53 mutations, the composite CR/PR rate was 80% (4/5). Two of these patients stayed on study for >10 cycles. Baseline bone marrow blast percentage or cytogenetic/molecular risk did not influence ORR. This study was registered at www.clinicaltrials.gov as #NCT01814826.

Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative neoplasms that are characterized by thrombohemorrhagic complications, symptom burden, and impaired survival mainly due to thrombosis, progression to myelofibrosis, and transformation to acute leukemia. In this manuscript, we will review the most recent changes in diagnostic criteria, the improvements in risk stratification, and the “state of the art” in the daily management of these disorders. The role of conventional therapies and novel agents, interferon α and the JAK2 inhibitor ruxolitinib, is critically discussed based on the results of a few basic randomized clinical studies. Several unmet needs remain, above all, the lack of a curative approach that might overcome the still burdensome morbidity and mortality of these hematologic neoplasms, as well as the toxicities associated with therapeutic agents.

The typical genome of chronic lymphocytic leukemia (CLL) carries ∼2000 molecular lesions. Few mutations recur across patients at a frequency >5%, whereas a large number of biologically and clinically uncharacterized genes are mutated at lower frequency. Approximately 80% of CLL patients carry at least 1 of 4 common chromosomal alterations, namely deletion 13q14, deletion 11q22-23, deletion 17p12, and trisomy 12. Knowledge of the CLL genome has translated into the availability of molecular biomarkers for prognosis and treatment prediction. Prognostic biomarkers do not affect treatment choice, and can be integrated into prognostic scores that are based on both clinical and biological variables. Molecular predictive biomarkers affect treatment choice, and currently include TP53 disruption by mutation and/or deletion and IGHV mutation status. TP53 disruption by gene mutation and/or deletion associates with chemoimmunotherapy failure and mandates treatment with innovative drugs, including ibrutinib, idelalisib, or venetoclax. The mutation status of IGHV genes represents a predictive biomarker for identifying patients that may benefit the most from chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab. Assessment of these biomarkers at the time of treatment requirement is recommended by most current guidelines for CLL management. Other molecular predictors are under investigation, but their application in clinical practice is premature.

Cardiovascular (CV) health has emerged as an important consideration in patients with chronic myeloid leukemia (CML) because of improved prognosis. Indeed, the success of BCR-ABL1 tyrosine kinase inhibitors (TKIs) has increased the focus on survivorship and late toxicity in oncological care. Survivorship issues in this population include CV disease prevention, given its prevalence in the general population. The introduction of BCR-ABL1 TKIs represented a unique concept of indefinite cancer therapy, only recently evolving to include “treatment-free remission.” Importantly, later-generation BCR-ABL1 TKIs have been associated with CV complications. Dasatinib has been associated with pleural/pericardial effusions and pulmonary hypertension, whereas nilotinib and ponatinib have been linked to the development of vascular occlusive events. There is currently a dearth of data with respect to the mechanisms of drug toxicities, the subsets of patients at risk, and prevention and treatment strategies to mitigate CV complications in patients with CML. Nevertheless, optimal patient CV risk assessment needs to become a more central tenet of patient care in CML. We propose several practical considerations for the practicing oncologist relative to the CV health of patients with CML, especially those on chronic TKI therapy.

Splenic marginal zone lymphoma (SMZL) and nodal marginal zone lymphoma (NMZL) are rare indolent chronic B-cell lymphomas. Prognosis is typically good with median survival around 10-15 years. Management is generally based on the presence of symptoms or high tumor burden. There are no standard treatments for these 2 entities, and therapeutic strategies are rapidly evolving. Clinical developments for these 2 entities are oriented by genomic studies, with largely overlapping mutational profiles involving the NOTCH, B-cell receptor (BcR) and nuclear factor κB (NF-κB) signaling, chromatin remodeling, and the cytoskeleton. Although new therapeutic options based on targeting signaling pathways and overcoming resistance are increasingly available, few specific prospective studies are performed for these rare subtypes, limiting the conclusions that can be drawn. Novel drugs targeting B-cell signaling have shown promise, including ibrutinib and copanlisib. The second-generation oral immunomodalator (IMiD) lenalidomide showed impressive results when combined with rituximab. Other potential solutions include targeting the NF-κB, JAK/STAT, BCL2, NOTCH, and Toll-like receptor signaling pathways; however, studies in these 2 MZL entities are yet to prove a definitive benefit. Molecular profiling is now a cornerstone of diagnostic, prognostic, and therapeutic strategies to offer patient- and disease-specific solutions. The development of a wider range of effective targeted therapies and prognostic biomarkers is keenly awaited and is expected to strongly affect the natural history of SMZL and NMZL.

Since its approval in 2011, the Janus kinase 1/2 (JAK1/2) inhibitor ruxolitinib has evolved to become the centerpiece of therapy for myelofibrosis (MF), and its use in patients with hydroxyurea resistant or intolerant polycythemia vera (PV) is steadily increasing. Several other JAK2 inhibitors have entered clinical testing, but none have been approved and many have been discontinued. Importantly, the activity of these agents is not restricted to patients with JAK2 V617F or exon 12 mutations. Although JAK2 inhibitors provide substantial clinical benefit, their disease-modifying activity is limited, and rational combinations with other targeted agents are needed, particularly in MF, in which survival is short. Many such combinations are being explored, as are other novel agents, some of which could successfully be combined with JAK2 inhibitors in the future. In addition, new JAK2 inhibitors with the potential for less myelosuppression continue to be investigated. Given the proven safety and efficacy of ruxolitinib, it is likely that ruxolitinib-based combinations will be a major way forward in drug development for MF. If approved, less myelosuppressive JAK2 inhibitors such as pacritinib or NS-018 could prove to be very useful additions to the therapeutic armamentarium in MF. In PV, inhibitors of histone deacetylases and human double minute 2 have activity, but their role, if any, in the future treatment algorithm is uncertain, given the availability of ruxolitinib and renewed interest in interferons. Ruxolitinib is in late-phase clinical trials in essential thrombocythemia, in which it could fill an important void for patients with troublesome symptoms.

Hemophagocytic lymphohistiocytosis (HLH) is an often-fatal disorder characterized by the overactivation of T cells and macrophages that excessively produce proinflammatory cytokines, including interferon-γ (IFN-γ). Previously, we reported that the JAK inhibitor ruxolitinib dampens T-cell activation and lessens inflammation in a model of HLH in which perforin-deficient (Prf1−/−) mice are infected with lymphocytic choriomeningitis virus (LCMV). Ruxolitinib inhibits signaling downstream of IFN-γ, as well as several other JAK-dependent cytokines. As a consequence, it remained unclear whether ruxolitinib was exerting its beneficial effects in HLH by inhibiting IFN-γ signaling or by targeting signaling initiated by other proinflammatory cytokines. To address this question, we compared the effects of ruxolitinib with those obtained using an IFN-γ–neutralizing antibody (αIFN-γ) in 2 murine HLH models. In both models, ruxolitinib and αIFN-γ reduced inflammation-associated anemia, indicating that ruxolitinib operates in an IFN-γ–dependent manner to reverse this HLH manifestation. In contrast, the number and activation status of T cells and neutrophils, as well as their infiltration into tissues, were significantly reduced following treatment with ruxolitinib, but they remained unchanged or were increased following treatment with αIFN-γ. Notably, despite discontinuation of ruxolitinib, LCMV-infected Prf1−/− mice exhibited enhanced survival compared with mice in which αIFN-γ was discontinued. This protective effect could be mimicked by transient treatment with αIFN-γ and a neutrophil-depleting antibody. Thus, ruxolitinib operates through IFN-γ–dependent and -independent mechanisms to dampen HLH by targeting the deleterious effects of T cells and neutrophils, with the latter representing an unappreciated and understudied cell type that contributes to HLH pathogenesis.

This phase 1/2 study assessed parsaclisib (INCB050465), a next-generation, potent, and highly selective phosphatidylinositol 3-kinase δ (PI3Kδ) inhibitor, in patients with relapsed or refractory B-cell malignancies, alone or in combination with a Janus kinase 1 inhibitor (itacitinib) or chemotherapy (rituximab, ifosfamide, carboplatin, and etoposide). Seventy-two patients received parsaclisib monotherapy (5-45 mg once daily). Expansion doses were 20 and 30 mg once daily; intermittent dosing at 20 mg (once daily for 9 weeks, then once weekly) was explored. No dose-limiting toxicities were identified, and maximum tolerated dose was not reached. Most common nonhematologic treatment-emergent adverse events (TEAEs) were diarrhea/colitis (36%), nausea (36%), fatigue (31%), and rash (31%). Grade 3/4 neutropenia occurred in 19% of patients. Serious TEAEs (>2 patients) were diarrhea/colitis (n = 9), pyrexia (n = 4), hypotension (n = 3), and sepsis (n = 3). Aspartate and alanine transaminase elevations occurring before treatment discontinuation were grade 1, except 1 grade 3 event each, secondary to sepsis. Two patients experienced 3 fatal parsaclisib-unrelated TEAEs (respiratory failure; respiratory failure and sepsis). In non-Hodgkin lymphoma (NHL), objective response rates to monotherapy were 71% in follicular lymphoma, 78% in marginal zone lymphoma, 67% in mantle cell lymphoma, and 30% in diffuse large B-cell lymphoma; 93% of responses occurred at first assessment (∼9 weeks). Parsaclisib has demonstrated antitumor activity in relapsed or refractory B-cell NHL with the potential for improved long-term patient outcomes. Phase 2 studies in relapsed or refractory B-cell NHL subtypes are ongoing. This trial was registered at www.clinicaltrials.gov as #NCT02018861.

Myeloproliferative neoplasms (MPN), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, have a propensity to evolve into accelerated and blast-phase disease (MPN-AP/BP), carrying a dismal prognosis. Conventional antileukemia therapy has limited efficacy in this setting. Thus, MPN-AP/BP is an urgent unmet clinical need. Modest responses to hypomethylating agents and single-agent ruxolitinib have been reported. More recently, combination of ruxolitinib and decitabine has demonstrated synergistic in vitro activity in human and murine systems. These observations led us to conduct a phase 1 study to explore the safety of combined decitabine and dose-escalated ruxolitinib in patients with MPN-AP/BP. A total of 21 patients were accrued to this multicenter study. Ruxolitinib was administered at doses of 10, 15, 25, or 50 mg twice daily in combination with decitabine (20 mg/m2 per day for 5 days) in 28-day cycles. The maximum tolerated dose was not reached. The most common reasons for study discontinuation were toxicity/adverse events (37%) and disease progression (21%). Fourteen patients died during study treatment period or follow-up. The median overall survival for patients on study was 7.9 months (95% confidence interval, 4.1-not reached). Among evaluable patients, the overall response rate by protocol-defined criteria (complete remission with incomplete count recovery + partial remission) was 9/17 (53%) and by intention-to-treat analysis was 9/21 (42.9%). The combination of decitabine and ruxolitinib was generally well tolerated by patients with MPN-AP/BP and demonstrates potentially promising clinical activity. A phase 2 trial evaluating the efficacy of this combination regimen is ongoing within the Myeloproliferative Disorder Research Consortium.

Although the therapy of chronic lymphocytic leukemia (CLL) has changed rapidly over the last 5 years, the key considerations in selecting a therapy for a previously treated patient with CLL continue to include the nature of the prior therapy and the duration of prior remission to that therapy, the prognostic features of the disease, and the health and comorbidities of the patient in question. For patients treated initially with chemoimmunotherapy, randomized trials have demonstrated the benefit of targeted therapy. Retrospective data suggest that ibrutinib is preferred as a first kinase inhibitor, whereas recent data with venetoclax and rituximab may challenge the choice of ibrutinib as a first novel agent in the relapsed setting. Data on sequencing of novel agents remain quite sparse, consisting of 1 prospective trial that demonstrated the efficacy of venetoclax in patients who have experienced progression with a kinase inhibitor, as well as a retrospective real-world analysis supporting this observation. Novel agents in advanced clinical development include primarily next-generation Bruton’s tyrosine kinase and phosphatidylinositol 3-kinase δ inhibitors, with other classes still in phase 1 trials. Clinical trials of combination time-limited therapies with the goal of deep remission and discontinuation are also in progress.

The treatment landscape of chronic lymphocytic leukemia (CLL) has changed dramatically in the last few years. The role of chemoimmunotherapy has declined significantly for patients with CLL. Fludarabine, cyclophosphamide, rituximab chemotherapy remains the standard frontline therapy for young fit patients with CLL, especially if IGHV mutated. For older adults, ibrutinib has been shown to be superior to chlorambucil. Hence, the role of chlorambucil monotherapy in the current era in the management of CLL is limited. The combination of chlorambucil and obinutuzumab is an alternative option for patients with comorbidities. For patients with del(17p), ibrutinib has become the standard treatment in the frontline setting. Several phase 3 trials with novel targeted agents, either as monotherapy or in combination, are either ongoing or have completed accrual. The results of many of these trials are expected in the next 1 to 2 years, and they will further help refine the frontline treatment strategy.

Hemophagocytic lymphohistiocytosis (HLH) comprises an emerging spectrum of inherited and noninherited disorders of the immune system characterized by the excessive production of cytokines, including interferon-γ and interleukins 2, 6, and 10 (IL-2, IL-6, and IL-10). The Janus kinases (JAKs) transduce signals initiated following engagement of specific receptors that bind a broad array of cytokines, including those overproduced in HLH. Based on the central role for cytokines in the pathogenesis of HLH, we sought to examine whether the inhibition of JAK function might lessen inflammation in murine models of the disease. Toward this end, we examined the effects of JAK inhibition using a model of primary (inherited) HLH in which perforin-deficient (Prf1(-∕-)) mice are infected with lymphocytic choriomeningitis virus (LCMV) and secondary (noninherited) HLH in which C57BL/6 mice receive repeated injections of CpG DNA. In both models, treatment with the JAK1/2 inhibitor ruxolitinib significantly lessened the clinical and laboratory manifestations of HLH, including weight loss, organomegaly, anemia, thrombocytopenia, hypercytokinemia, and tissue inflammation. Importantly, ruxolitinib treatment also significantly improved the survival of LCMV-infectedPrf1(-∕-)mice. Mechanistic studies revealed that in vivo exposure to ruxolitinib inhibited signal transducer and activation of transcription 1-dependent gene expression, limited CD8(+)T-cell expansion, and greatly reduced proinflammatory cytokine production, without effecting degranulation and cytotoxic function. Collectively, these findings highlight the JAKs as novel, druggable targets for mitigating the cytokine-driven hyperinflammation that occurs in HLH. These observations also support the incorporation of JAK inhibitors such as ruxolitinib into future clinical trials for patients with these life-threatening disorders.

Mantle cell lymphoma (MCL) is characterized by an aggressive clinical course and inevitable development of refractory disease, stressing the need to develop alternative therapeutic strategies. To this end, we evaluated pevonedistat (MLN4924), a novel potent and selective NEDD8-activating enzyme inhibitor in a panel of MCL cell lines, primary MCL tumor cells, and 2 distinct murine models of human MCL. Pevonedistat exposure resulted in a dose-, time-, and caspase-dependent cell death in the majority of the MCL cell lines and primary tumor cells tested. Of interest, in the MCL cell lines with lower half-maximal inhibitory concentration (0.1-0.5 μM), pevonedistat induced G1-phase cell cycle arrest, downregulation of Bcl-xL levels, decreased nuclear factor (NF)-κB activity, and apoptosis. In addition, pevonedistat exhibited additive/synergistic effects when combined with cytarabine, bendamustine, or rituximab. In vivo, as a single agent, pevonedistat prolonged the survival of 2 MCL-bearing mouse models when compared with controls. Pevonedistat in combination with rituximab led to improved survival compared with rituximab or pevonedistat monotherapy. Our data suggest that pevonedistat has significant activity in MCL preclinical models, possibly related to effects on NF-κB activity, Bcl-xL downregulation, and G1 cell cycle arrest. Our findings support further investigation of pevonedistat with or without rituximab in the treatment of MCL.

Myelofibrosis (MF) is complex at the pathobiologic level and heterogeneous at the clinical level. The advances in molecular characterization of MF provide important insight into the mechanisms driving this chronic myeloid malignancy, refine risk stratification, offer novel therapeutic targets, and serve to measure therapeutic response. Although JAK2 inhibition has been the focus of laboratory and clinical efforts over the last decade, current experimental therapeutic approaches have broadened to include inhibitors of key alternative signaling pathways, epigenetic modulators, anti-fibrotics, and immunotherapies. Based on compelling preclinical rationale, a number of JAK2 inhibitor based combination therapies are now actively being evaluated in the clinic with the goal of disease course modification. The role and timing of hematopoietic stem cell transplant (HSCT) for MF has been challenged with the availability of commercial ruxolitinib and the plethora of experimental treatment options that exist. Integration of preconditioning JAK2 inhibition, reduced intensity conditioning regimens, and alternative donor sources are all being explored in an attempt to optimize this potentially curative modality. This review will summarize modern MF risk stratification, current clinical research approaches to chronic and advance phase MF focusing on novel agents alone and in combination, and update the reader on new directions in HSCT.

Ibrutinib is a novel oral therapy that has shown significant efficacy as initial treatment of chronic lymphocytic leukemia (CLL). It is a high-cost continuous therapy differing from other regimens that are given for much shorter courses. Our objective was to evaluate the cost-effectiveness of ibrutinib for first-line treatment of CLL in patients older than age 65 years without a 17p deletion. We developed a semi-Markov model to analyze the cost-effectiveness of ibrutinib vs a comparator therapy from a US Medicare perspective. No direct comparison between ibrutinib and the best available treatment alternative, obinutuzumab plus chlorambucil (chemoimmunotherapy), exists. Therefore, we compared ibrutinib to a theoretical treatment alternative, which was modeled to confer the effectiveness of an inferior treatment (chlorambucil alone) and the costs and adverse events of chemoimmunotherapy, which would provide ibrutinib with the best chance of being cost-effective. Even so, the incremental cost-effectiveness ratio of ibrutinib vs the modeled comparator was $189 000 per quality-adjusted life-year (QALY) gained. To reach a willingness-to-pay threshold (WTP) of $150 000 per QALY, the monthly cost of ibrutinib would have to be at most $6800, $1700 less than the modeled cost of $8500 per month (a reduction of $20 400 per year). When the comparator efficacy is increased to more closely match that seen in trials evaluating chemoimmunotherapy, ibrutinib costs more than $262 000 per QALY gained, and the monthly cost of ibrutinib would need to be lowered to less than $5000 per month to be cost-effective. Ibrutinib is not cost-effective as initial therapy at a WTP threshold of $150 000 per QALY gained.

The Bruton tyrosine kinase inhibitor ibrutinib induces high rates of clinical response in chronic lymphocytic leukemia (CLL). However, there remains a need for adjunct treatments to deepen response and to overcome drug resistance. Blinatumomab, a CD19/CD3 bispecific antibody (bsAb) designed in the BiTE (bispecific T-cell engager) format, is approved by the US Food and Drug Administration for the treatment of relapsed or refractory B-cell precursor acute lymphoblastic leukemia. Because of its short half-life of 2.1 hours, blinatumomab requires continuous intravenous dosing for efficacy. We developed a novel CD19/CD3 bsAb in the single-chain Fv-Fc format (CD19/CD3-scFv-Fc) with a half-life of ∼5 days. In in vitro experiments, both CD19/CD3-scFv-Fc and blinatumomab induced >90% killing of CLL cells from treatment-naive patients. Antileukemic activity was associated with increased autologous CD8 and CD4 T-cell proliferation, activation, and granzyme B expression. In the NOD/SCID/IL2Rγnull patient-derived xenograft mouse model, once-weekly treatment with CD19/CD3-scFv-Fc eliminated >98% of treatment-naive CLL cells in blood and spleen. By contrast, blinatumomab failed to induce a response, even when administered daily. We next explored the activity of CD19/CD3-scFv-Fc in the context of ibrutinib treatment and ibrutinib resistance. CD19/CD3-scFv-Fc induced more rapid killing of CLL cells from ibrutinib-treated patients than those from treatment-naive patients. CD19/CD3-scFv-Fc also demonstrated potent activity against CLL cells from patients with acquired ibrutinib-resistance harboring BTK and/or PLCG2 mutations in vitro and in vivo using patient-derived xenograft models. Taken together, these data support investigation of CD19/CD3 bsAb’s and other T cell-recruiting bsAb’s as immunotherapies for CLL, especially in combination with ibrutinib or as rescue therapy in ibrutinib-resistant disease.

Ibrutinib, an oral covalent inhibitor of Bruton’s tyrosine kinase, is an effective therapy for patients with chronic lymphocytic leukemia (CLL). To determine whether rituximab provides added benefit to ibrutinib, we conducted a randomized single-center trial of ibrutinib vs ibrutinib plus rituximab. Patients with CLL requiring therapy were randomized to receive 28-day cycles of once-daily ibrutinib 420 mg, either as a single agent (n = 104), or together with rituximab (375 mg/m(2); n = 104), given weekly during cycle 1, then once per cycle until cycle 6. The primary end point was progression-free survival (PFS) in the intention-to-treat population. We enrolled 208 patients with CLL, 181 with relapsed CLL and 27 treatment-naive patients with high-risk disease (17p deletion or TP53 mutation). After a median follow-up of 36 months, the Kaplan-Meier estimates of PFS were 86% (95% confidence interval [CI], 76.6-91.9) for patients receiving ibrutinib, and 86.9% (95% CI, 77.3-92.6) for patients receiving ibrutinib plus rituximab. Similarly, response rates were the same in both arms (overall response rate, 92%). However, time to normalization of peripheral blood lymphocyte counts and time to complete remission were shorter, and residual disease levels in the bone marrow were lower, in patients receiving ibrutinib plus rituximab. We conclude that the addition of rituximab to ibrutinib in relapsed and treatment-naive high-risk patients with CLL failed to show improvement in PFS. However, patients treated with ibrutinib plus rituximab reached their remissions faster and achieved significantly lower residual disease levels. Given these results, ibrutinib as single-agent therapy remains current standard-of-care treatment in CLL. This trial was registered at www.clinicaltrials.gov as #NCT02007044.

Myeloproliferative neoplasms (MPNs) often carry JAK2(V617F), MPL(W515L), or CALR(del52) mutations. Current treatment options for MPNs include cytoreduction by hydroxyurea and JAK1/2 inhibition by ruxolitinib, both of which are not curative. We show here that cell lines expressing JAK2(V617F), MPL(W515L), or CALR(del52) accumulated reactive oxygen species-induced DNA double-strand breaks (DSBs) and were modestly sensitive to poly-ADP-ribose polymerase (PARP) inhibitors olaparib and BMN673. At the same time, primary MPN cell samples from individual patients displayed a high degree of variability in sensitivity to these drugs. Ruxolitinib inhibited 2 major DSB repair mechanisms, BRCA-mediated homologous recombination and DNA-dependent protein kinase-mediated nonhomologous end-joining, and, when combined with olaparib, caused abundant accumulation of toxic DSBs resulting in enhanced elimination of MPN primary cells, including the disease-initiating cells from the majority of patients. Moreover, the combination of BMN673, ruxolitinib, and hydroxyurea was highly effective in vivo against JAK2(V617F)+ murine MPN-like disease and also against JAK2(V617F)+, CALR(del52)+, and MPL(W515L)+ primary MPN xenografts. In conclusion, we postulate that ruxolitinib-induced deficiencies in DSB repair pathways sensitized MPN cells to synthetic lethality triggered by PARP inhibitors.

The JAK2 c.1849G>T (p.V617F) mutation leads to constitutive activation of Janus kinase (JAK)2 and contributes to dysregulated JAK signaling in myelofibrosis (MF), polycythemia vera (PV), and essential thrombocythemia (ET). In the phase 3 Controlled Myelofibrosis Study with Oral JAK Inhibitor Treatment-I trial, patients with MF, post-PV MF, or post-ET MF achieved significant reductions in splenomegaly and improvements in symptoms with ruxolitinib vs placebo at week 24. This long-term follow-up analysis was performed to determine whether ruxolitinib therapy altered the JAK2p.V617F allele burden in JAK2p.V617F-positive patients. Assessments at baseline and weeks 24, 48, 120, 144, 168, and 216 demonstrated reductions in allele burden from baseline with ruxolitinib treatment that correlated with spleen volume reductions. Of 236 JAK2p.V617F-positive patients analyzed, 20 achieved partial and 6 achieved complete molecular responses, with median times to response of 22.2 and 27.5 months, respectively. Allele burden reductions were greater in patients with shorter disease duration, which suggests a potential benefit of earlier treatment. This trial was registered at www.clinicaltrials.gov as #NCT00952289.

We investigated the effects of nilotinib plus multiagent chemotherapy, followed by consolidation/maintenance or allogeneic hematopoietic cell transplantation (allo-HCT) for adult patients with newly diagnosed Philadelphia-positive (Ph-pos) acute lymphoblastic leukemia (ALL). Study subjects received induction treatment that comprised concurrent vincristine, daunorubicin, prednisolone, and nilotinib. After achieving complete hematologic remission (HCR), subjects received either 5 courses of consolidation, followed by 2-year maintenance with nilotinib, or allo-HCT. Minimal residual disease (MRD) was assessed at HCR, and every 3 months thereafter. The molecular responses (MRs) were defined as MR3 for BCR-ABL1/G6PDH ratios ≤10(-3) and MR5 for ratios

Although most patients with myelofibrosis (MF) derive benefit from ruxolitinib, some are refractory, have a suboptimal response, or quickly lose their response. To identify genes that may predict response to ruxolitinib, we performed targeted next-generation sequencing (NGS) of a panel of 28 genes recurrently mutated in hematologic malignancies in a cohort of patients with MF who were treated with ruxolitinib in a phase 1/2 study. We also tested for CALR deletions by standard polymerase chain reaction methods. Ninety-eight percent of patients had a mutation in ≥1 gene. Seventy-nine (82.1%) patients had the JAK2(V617F) mutation, 9 (9.5%) had CALR mutations (7 type 1, 2 type 2), 3 (3.1%) had MPL mutations, and 4 (4.2%) were negative for all 3. ASXL1/JAK2 and TET2/JAK2 were the most frequently comutated genes. Mutations in NRAS, KRAS, PTPN11, GATA2, TP53, and RUNX1 were found in

Novel therapies for chronic graft-versus-host disease (cGVHD) are needed. Aberrant B-cell activation has been demonstrated in mice and humans with cGVHD. Having previously found that human cGVHD B cells are activated and primed for survival, we sought to further evaluate the role of the spleen tyrosine kinase (Syk) in cGVHD in multiple murine models and human peripheral blood cells. In a murine model of multiorgan system, nonsclerodermatous disease with bronchiolitis obliterans where cGVHD is dependent on antibody and germinal center (GC) B cells, we found that activation of Syk was necessary in donor B cells, but not T cells, for disease progression. Bone marrow-specific Syk deletion in vivo was effective in treating established cGVHD, as was a small-molecule inhibitor of Syk, fostamatinib, which normalized GC formation and decreased activated CD80/86(+) dendritic cells. In multiple distinct models of sclerodermatous cGVHD, clinical and pathological disease manifestations were not eliminated when mice were therapeutically treated with fostamatinib, though both clinical and immunologic effects could be observed in one of these scleroderma models. We further demonstrated that Syk inhibition was effective at inducing apoptosis of human cGVHD B cells. Together, these data demonstrate a therapeutic potential of targeting B-cell Syk signaling in cGVHD.

In this study, we randomly compared high doses of the tyrosine kinase inhibitor imatinib combined with reduced-intensity chemotherapy (arm A) to standard imatinib/hyperCVAD (cyclophosphamide/vincristine/doxorubicin/dexamethasone) therapy (arm B) in 268 adults (median age, 47 years) with Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL). The primary objective was the major molecular response (MMolR) rate after cycle 2, patients being then eligible for allogeneic stem cell transplantation (SCT) if they had a donor, or autologous SCT if in MMolR and no donor. With fewer induction deaths, the complete remission (CR) rate was higher in arm A than in arm B (98% vs 91%; P = .006), whereas the MMolR rate was similar in both arms (66% vs 64%). With a median follow-up of 4.8 years, 5-year event-free survival and overall survival (OS) rates were estimated at 37.1% and 45.6%, respectively, without difference between the arms. Allogeneic transplantation was associated with a significant benefit in relapse-free survival (hazard ratio [HR], 0.69; P = .036) and OS (HR, 0.64; P = .02), with initial white blood cell count being the only factor significantly interacting with this SCT effect. In patients achieving MMolR, outcome was similar after autologous and allogeneic transplantation. This study validates an induction regimen combining reduced-intensity chemotherapy and imatinib in Ph+ ALL adult patients and suggests that SCT in first CR is still a good option for Ph+ ALL adult patients. This trial was registered at www.clinicaltrials.gov as #NCT00327678.

Pacritinib (SB1518) is a Janus kinase 2 (JAK2), JAK2(V617F), and Fms-like tyrosine kinase 3 inhibitor that does not inhibit JAK1. It demonstrated a favorable safety profile with promising efficacy in phase 1 studies in patients with primary and secondary myelofibrosis (MF). This multicenter phase 2 study further characterized the safety and efficacy of pacritinib in the treatment of patients with MF. Eligible patients had clinical splenomegaly poorly controlled with standard therapies or were newly diagnosed with intermediate- or high-risk Lille score. Patients with any degree of cytopenia were eligible. Thirty-five patients were enrolled. At entry, 40% had hemoglobin

Ibrutinib is a Bruton tyrosine kinase inhibitor approved for the treatment of patients with relapsed refractory chronic lymphocytic leukemia (RR-CLL). We describe the characteristics, causes of discontinuation, and outcomes in patients who discontinued treatment with ibrutinib. One hundred twenty-seven patients were enrolled in various clinical trials of ibrutinib, with or without rituximab, at our center. Thirty-three (26%) patients have discontinued ibrutinib to date. The majority of those patients had high-risk features: 94% with unmutated immunoglobulin heavy chain variable gene rearrangement, 58% with del(17p) by fluorescence in situ hybridization, and 54% with a complex karyotype. Causes of discontinuation were disease transformation (7), progressive CLL (7), stem cell transplantation (3), adverse events (11), serious adverse events/deaths (3), and miscellaneous reasons (2). Twenty five patients (76%) died after discontinuing ibrutinib; the median overall survival was 3.1 months after discontinuation. Most patients with RR-CLL who discontinued ibrutinib early were difficult to treat and had poor outcomes.

Early T-cell precursor (ETP) acute lymphoblastic leukemia (ALL) is a recently described subtype of T-ALL characterized by a unique immunophenotype and genomic profile, as well as a high rate of induction failure. Frequent mutations in cytokine receptor and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways led us to hypothesize that ETP-ALL is dependent on JAK/STAT signaling. Here we demonstrate aberrant activation of the JAK/STAT pathway in ETP-ALL blasts relative to non-ETP T-ALL. Moreover, ETP-ALL showed hyperactivation of STAT5 in response to interleukin-7, an effect that was abrogated by the JAK1/2 inhibitor ruxolitinib. In vivo, ruxolitinib displayed activity in 6 of 6 patient-derived murine xenograft models of ETP-ALL, with profound single-agent efficacy in 5 models. Ruxolitinib treatment decreased peripheral blast counts relative to pretreatment levels and compared with control (P < .01) in 5 of 6 ETP-ALL xenografts, with marked reduction in mean splenic blast counts (P < .01) in 6 of 6 samples. Surprisingly, both JAK/STAT pathway activation and ruxolitinib efficacy were independent of the presence of JAK/STAT pathway mutations, raising the possibility that the therapeutic potential of ruxolitinib in ETP-ALL extends beyond those cases with JAK mutations. These findings establish the preclinical in vivo efficacy of ruxolitinib in ETP-ALL, a biologically distinct subtype for which novel therapies are needed.

To the editor: Ibrutinib, an orally bioavailable Bruton tyrosine kinase (BTK) inhibitor,1 has shown significant clinical benefit for chronic lymphocytic leukemia (CLL) patients.2,3 However, ibrutinib monotherapy often leads to partial responses and extended lymphocytosis,4 which led us to question whether combination therapy could provide complete clinical responses. To identify pharmacologic agents that complement ibrutinib therapy, we performed an ex vivo drug screen using targeted agents on CLL cells isolated from ibrutinib-treated patients (Table 1). We identified carfilzomib (PR-171), a second-generation proteasome inhibitor5 and ABT-1996 (a Bcl-2 antagonist) as the most cytotoxic agents, as indicated by increased annexin V/propidium iodide (PI) double positivity after 24-hour incubation (Figure 1A7). Results were consistent in samples from 7 additional patients (data not shown). Carfilzomib cytotoxic effect after 24-hour treatment was confirmed in samples from 23 patients treated with ibrutinib for 2 to 23 weeks (Figure 1B). Table 1 Patient characteristics Figure 1 Biological and molecular consequences of adding CFZ to IBT in primary CLL lymphocytes. Peripheral blood was obtained from CLL patients who were either IBT-naive or undergoing IBT therapy and who had given written informed consent in accordance with the ... Next, we evaluated carfilzomib and ibrutinib synergetic cytotoxic effect on ibrutinib-naive CLL cells during in vitro incubation. Cells isolated from 7 patients (Table 1) were treated for 16 hours with concentrations of ibrutinib and carfilzomib that resulted in modest toxicity individually but, when combined, showed at least an additive cytotoxic effect (Figure 1C). The response to single agents and the combination varied between patients. Western blot analysis of CLL cells isolated from 2 patients confirmed the inhibitory effect of carfilzomib on the proteasome machinery, as illustrated by the accumulation of polyubiquitinated proteins and the stabilization of a short-lived protein (ie, p-IκBα) in a dose-dependent manner (Figure 1D8). Furthermore, carfilzomib induced a dose-dependent activation of caspase 3 and accumulation of cleaved PARP and Bcl-2 (Figure 1D), both of which were consistent with a previous study where CLL samples with 17p del also responded well to the cytotoxic effect of carfilzomib.9 Ibrutinib treatment resulted in decreased p-BTK at tyr-223, with a minor effect on caspase activation; however, when combined with carfilzomib, an enhancement of cleaved caspase 3 was notably observed with low doses of carfilzomib (Figure 1D). Notably, increasing concentrations of carfilzomib caused accumulation of the transcription factor CHOP and the proapoptotic BH3-only protein Noxa (Figure 1D), which is consistent with evidence that proteasome inhibition leads to activation of the endoplasmic reticulum stress response and accumulation of apoptotic regulators.10,11 Interestingly, Noxa was previously shown to play a critical role in bortezomib-induced apoptosis in CLL; however, treatment with this reversible inhibitor did not result in increased of CHOP protein,12 suggesting different modes of action between bortezomib and carfilzomib. Here, ibrutinib cotreatment did not influence carfilzomib’s ability to induce accumulation of polyubiquitinated proteins, p-IκBα, Noxa, and CHOP (Figure 1D). To compare carfilzomib-induced cytotoxic responses between untreated and ibrutinib-treated samples, we isolated CLL cells from 8 patients prior to and 4 weeks after the initiation of ibrutinib therapy, which was previously reported as the time line for maximum transient lymphocytosis.2 With ibrutinib therapy, 4 patients exhibited signs of lymphocytosis as previously reported2: 2 remained unchanged and 2 patients showed a >80% decrease in absolute lymphocyte count (ALC) (Figure 1E). No notable differences were observed in carfilzomib responses between CLL cells isolated before and after ibrutinib treatment, except 3 patients (CLL-967, CLL-864, and CLL-488) whose cells showed increased sensitivity to carfilzomib in ibrutinib-treated samples (Figure 1F). In contrast, the ibrutinib-treated sample from patient CLL-683 had a marked decrease in apoptosis (Figure 1F), possibly due to a >80% reduction in his ALC (Figure 1E), and similar results were observed in patient CLL-826, 12 weeks following ibrutinib treatment (Figure 1E). These observations are consistent with the report that carfilzomib is less cytotoxic to normal lymphocytes than CLL cells.9 We next used western blot analysis to assess the effects of carfilzomib treatment on CLL cells isolated before and after ibrutinib treatment from 2 patients, 1 with therapy-related lymphocytosis (CLL-967) and 1 without (CLL-630). Cells from both patients responded in a similar manner to carfilzomib treatment, irrespective of ibrutinib therapy, as indicated by the accumulation of polyubiquitinated proteins, p-IκBα, CHOP, and Noxa (Figure 1G). Additionally, carfilzomib treatment in both untreated and ibrutinib-treated cells showed caspase 3 activation and accumulation of cleaved PARP and Bcl-2 (Figure 1G). We extended these findings with cells isolated from 3 patients before and after 2, 4, or 12 weeks of ibrutinib treatment, respectively; in all cases, the cells exhibited decreased phosphorylation of BTK (Figure 1H). Furthermore, both the extrinsic and the intrinsic apoptotic pathways were activated by carfilzomib treatment, as indicated by increases in cleaved caspase 8 and caspase 9, respectively (Figure 1H), and to some extent the cells appeared more resistant to spontaneous endogenous apoptosis ex vivo following ibrutinib therapy. In summary, this pilot study provides some foundation to further investigate carfilzomib-ibrutinib combination therapy for CLL.

Myelofibrosis (MF) is a BCR-ABL1–negative myeloproliferative neoplasm characterized by clonal myeloproliferation, dysregulated kinase signaling, and release of abnormal cytokines. In recent years, important progress has been made in the knowledge of the molecular biology and the prognostic assessment of MF. Conventional treatment has limited impact on the patients’ survival; it includes a wait-and-see approach for asymptomatic patients, erythropoiesis-stimulating agents, androgens, or immunomodulatory agents for anemia, cytoreductive drugs such as hydroxyurea for the splenomegaly and constitutional symptoms, and splenectomy or radiotherapy in selected patients. The discovery of the Janus kinase (JAK)2 mutation triggered the development of molecular targeted therapy of MF. The JAK inhibitors are effective in both JAK2-positive and JAK2-negative MF; one of them, ruxolitinib, is the current best available therapy for MF splenomegaly and constitutional symptoms. However, although ruxolitinib has changed the therapeutic scenario of MF, there is no clear indication of a disease-modifying effect. Allogeneic stem cell transplantation remains the only curative therapy of MF, but due to its associated morbidity and mortality, it is usually restricted to eligible high- and intermediate-2–risk MF patients. To improve current therapeutic results, the combination of JAK inhibitors with other agents is currently being tested, and newer drugs are being investigated.

Chronic myeloid leukemia (CML) is a stem cell (SC) neoplasm characterized by the BCR/ABL1 oncogene. Although mechanisms of BCR/ABL1-induced transformation are well-defined, little is known about effector-molecules contributing to malignant expansion and the extramedullary spread of leukemic SC (LSC) in CML. We have identified the cytokine-targeting surface enzyme dipeptidylpeptidase-IV (DPPIV/CD26) as a novel, specific and pathogenetically relevant biomarker of CD34(+)/CD38(─) CML LSC. In functional assays, CD26 was identified as target enzyme disrupting the SDF-1-CXCR4-axis by cleaving SDF-1, a chemotaxin recruiting CXCR4(+) SC. CD26 was not detected on normal SC or LSC in other hematopoietic malignancies. Correspondingly, CD26(+) LSC decreased to low or undetectable levels during successful treatment with imatinib. CD26(+) CML LSC engrafted NOD-SCID-IL-2Rγ(-/-) (NSG) mice with BCR/ABL1(+) cells, whereas CD26(─) SC from the same patients produced multilineage BCR/ABL1(-) engraftment. Finally, targeting of CD26 by gliptins suppressed the expansion of BCR/ABL1(+) cells. Together, CD26 is a new biomarker and target of CML LSC. CD26 expression may explain the abnormal extramedullary spread of CML LSC, and inhibition of CD26 may revert abnormal LSC function and support curative treatment approaches in this malignancy.

Graft-versus-host-disease (GVHD) is a severe complication of allogeneic hematopoietic cell transplantation (allo-HCT) characterized by the production of high levels of proinflammatory cytokines. Activated Janus kinases (JAKs) are required for T-effector cell responses in different inflammatory diseases, and their blockade could potently reduce acute GVHD. We observed that inhibition of JAK1/2 signaling resulted in reduced proliferation of effector T cells and suppression of proinflammatory cytokine production in response to alloantigen in mice. In vivo JAK 1/2 inhibition improved survival of mice developing acute GVHD and reduced histopathological GVHD grading, serum levels of proinflammatory cytokines, and expansion of alloreactive luc-transgenic T cells. Mechanistically, we could show that ruxolitinib impaired differentiation of CD4(+) T cells into IFN-γ- and IL17A-producing cells, and that both T-cell phenotypes are linked to GVHD. Conversely, ruxolitinib treatment in allo-HCT recipients increased FoxP3(+) regulatory T cells, which are linked to immunologic tolerance. Based on these results, we treated 6 patients with steroid-refractory GVHD with ruxolitinib. All patients responded with respect to clinical GVHD symptoms and serum levels of proinflammatory cytokines. In summary, ruxolitinib represents a novel targeted approach in GVHD by suppression of proinflammatory signaling that mediates tissue damage and by promotion of tolerogenic Treg cells.