Search

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.

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.

Signaling through JAK1 and/or JAK2 is common among tumor and nontumor cells within peripheral T-cell lymphoma (PTCL). No oral therapies are approved for PTCL, and better treatments for relapsed/refractory disease are urgently needed. We conducted a phase 2 study of the JAK1/2 inhibitor ruxolitinib for patients with relapsed/refractory PTCL (n = 45) or mycosis fungoides (MF) (n = 7). Patients enrolled onto 1 of 3 biomarker-defined cohorts: (1) activating JAK and/or STAT mutations, (2) ≥30% pSTAT3 expression among tumor cells by immunohistochemistry, or (3) neither or insufficient tissue to assess. Patients received ruxolitinib 20 mg PO twice daily until progression and were assessed for response after cycles 2 and 5 and every 3 cycles thereafter. The primary endpoint was clinical benefit rate (CBR), defined as the combination of complete response, partial response (PR), and stable disease lasting at least 6 months. Only 1 of 7 patients with MF had CBR (ongoing PR > 18 months). CBR among the PTCL cases (n = 45) in cohorts 1, 2, and 3 were 53%, 45%, and 13% (cohorts 1 & 2 vs 3, P = .02), respectively. Eight patients had CBR > 12 months (5 ongoing), including 4 of 5 patients with T-cell large granular lymphocytic leukemia. In an exploratory analysis using multiplex immunofluorescence, expression of phosphorylated S6, a marker of PI3 kinase or mitogen-activated protein kinase activation, in

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

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.

Some patients with paroxysmal nocturnal hemoglobinuria (PNH) remain persistently anemic despite treatment with eculizumab and may have significant extravascular hemolysis. Kulasekararaj and colleagues report a phase 2 trial of danicopan, a first-inclass oral complement factor D inhibitor, added to ongoing eculizumab therapy in PNH patients who still needed transfusions. This small study of 12 patients reveals that the combination has acceptable safety while substantially increasing the hemoglobin concentration, reducing extravascular hemolysis, and improving the fatigue score., Key Points Danicopan, a first-in-class oral factor D inhibitor showed clinical benefit when given in addition to eculizumab in patients with PNH.Addition of danicopan reduced extravascular hemolysis and improved anemia in patients with PNH with suboptimal eculizumab response., Visual Abstract, Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by uncontrolled terminal complement activation and subsequent intravascular hemolysis (IVH). C5 inhibitors prevent membrane attack complex formation, but patients may experience extravascular hemolysis (EVH) and continue to require blood transfusions. Danicopan, an oral proximal complement inhibitor of alternative pathway factor D (FD), is designed to control IVH and EVH. In a phase 2 dose-finding trial, eculizumab-treated transfusion-dependent patients with PNH (n = 12) received danicopan, 100 to 200 mg thrice daily, in addition to their eculizumab regimen for 24 weeks. End points included hemoglobin (Hgb) change vs baseline at week 24 (primary), reduction in blood transfusions, and patient-reported outcomes. Safety, tolerability, and pharmacokinetics/pharmacodynamics were measured. Twelve patients received ≥1 danicopan dose; 1 patients discontinued from a serious adverse event deemed unlikely related to danicopan. Eleven patients completed the 24-week treatment period. Addition of danicopan resulted in a mean Hgb increase of 2.4 g/dL at week 24. In the 24 weeks prior to danicopan, 10 patients received 31 transfusions (50 units) compared with 1 transfusion (2 units) in 1 patient during the 24-week treatment period. Mean Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue score increased by 11 points from baseline to week 24. The most common adverse events were headache, cough, and nasopharyngitis. Addition of danicopan, a first-in-class FD inhibitor, led to a meaningful improvement in Hgb and reduced transfusion requirements in patients with PNH who were transfusion-dependent on eculizumab. These benefits were associated with improvement of FACIT-Fatigue. This trial was registered at www.clinicaltrials.gov as #NCT03472885.

Approximately 80% of adult patients with immune thrombocytopenia (ITP) have treatment failure with corticosteroids or become dependent on them and require second-line therapy. Several new and effective therapies have been introduced during the past decade and our understanding of disease burden and its effect on quality of life has expanded. It is now recommended that splenectomy, the standard second-line therapy for decades, be delayed for at least 12 to 24 months, allowing for more patients to achieve remission on medical therapies before considering surgery. It is highly recommended that medical therapies be used that have abundant clinical trial evidence, such as the thrombopoietin receptor agonists (TPO-RAs) rituximab and fostamatinib. Unfortunately, there are no reliable biomarkers that help in treatment selection. These therapeutic medical options have variable efficacy, safety profiles, mechanisms of action, and modes of administration. This enables and mandates an individualized approach to treatment, where patient involvement, preferences and values have become central to the process of choosing the appropriate therapy. Both TPO-RAs and fostamatinib are maintenance therapies, whereas rituximab is given for a limited number of doses. Although the response is usually maintained while receiving a TPO-RA or fostamatinib therapy, half of rituximab responders will no longer respond 1 to 2 years after administration and require retreatment or other therapy.

Graft-versus-host disease (GVHD) remains a major limitation of allogeneic hematopoietic stem cell transplantation. Only half of patients with severe acute GVHD respond to first-line treatment with corticosteroids and, for several decades, there was no optimal second-line treatment of patients with corticosteroid-refractory acute GVHD. Ruxolitinib was recently approved for the treatment of corticosteroid-refractory acute GVHD in adult and pediatric patients 12 years and older. Thus, it is important to define the patient population that would now be considered as refractory to ruxolitinib vs ruxolitinib dependent. Here, we propose to define ruxolitinib-refractory acute GVHD as disease that shows: (1) progression of GVHD compared with baseline after at least 5 to 10 days of treatment with ruxolitinib, based either on objective increase in stage/grade, or new organ involvement; (2) lack of improvement in GVHD (partial response or better) compared with baseline after ≥14 days of treatment with ruxolitinib; or (3) loss of response, defined as objective worsening of GVHD determined by increase in stage, grade, or new organ involvement at any time after initial improvement. GVHD manifestations that persist without improvement in patients who had a grade ≥3 treatment-emergent and ruxolitinib-attributed adverse event that did not resolve within 7 days of discontinuing ruxolitinib would serve as a clinical indication for additional treatment. In addition, absence of complete response or very good partial response at day 28 after ruxolitinib could be considered as an eligibility criterion.

CRLF2-rearranged (CRLF2r) acute lymphoblastic leukemia (ALL) accounts for more than half of Philadelphia chromosome-like (Ph-like) ALL and is associated with a poor outcome in children and adults. Overexpression of CRLF2 results in activation of Janus kinase (JAK)-STAT and parallel signaling pathways in experimental models, but existing small molecule inhibitors of JAKs show variable and limited efficacy. Here, we evaluated the efficacy of proteolysis-targeting chimeras (PROTACs) directed against JAKs. Solving the structure of type I JAK inhibitors ruxolitinib and baricitinib bound to the JAK2 tyrosine kinase domain enabled the rational design and optimization of a series of cereblon (CRBN)-directed JAK PROTACs utilizing derivatives of JAK inhibitors, linkers, and CRBN-specific molecular glues. The resulting JAK PROTACs were evaluated for target degradation, and activity was tested in a panel of leukemia/lymphoma cell lines and xenograft models of kinase-driven ALL. Multiple PROTACs were developed that degraded JAKs and potently killed CRLF2r cell lines, the most active of which also degraded the known CRBN neosubstrate GSPT1 and suppressed proliferation of CRLF2r ALL in vivo, e.g. compound 7 (SJ988497). Although dual JAK/GSPT1-degrading PROTACs were the most potent, the development and evaluation of multiple PROTACs in an extended panel of xenografts identified a potent JAK2-degrading, GSPT1-sparing PROTAC that demonstrated efficacy in the majority of kinase-driven xenografts that were otherwise unresponsive to type I JAK inhibitors, e.g. compound 8 (SJ1008030). Together, these data show the potential of JAK-directed protein degradation as a therapeutic approach in JAK-STAT–driven ALL and highlight the interplay of JAK and GSPT1 degradation activity in this context.

Patients who develop steroid-refractory acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic cell transplantation have poor prognosis, highlighting an unmet therapeutic need. In this open-label phase 2 study (ClinicalTrials.gov identifier: NCT02953678), patients aged at least 12 years with grades II to IV steroid-refractory aGVHD were eligible to receive ruxolitinib orally, starting at 5 mg twice daily plus corticosteroids, until treatment failure, unacceptable toxicity, or death. The primary end point was overall response rate (ORR) at day 28; the key secondary end point was duration of response (DOR) at 6 months. As of 2 July 2018, 71 patients received at least 1 dose of ruxolitinib. Forty-eight of those patients (67.6%) had grade III/IV aGVHD at enrollment. At day 28, 39 patients (54.9%; 95% confidence interval, 42.7%-66.8%) had an overall response, including 19 (26.8%) with complete responses. Best ORR at any time was 73.2% (complete response, 56.3%). Responses were observed across skin (61.1%), upper (45.5%) and lower (46.0%) gastrointestinal tract, and liver (26.7%). Median DOR was 345 days. Overall survival estimate at 6 months was 51.0%. At day 28, 24 (55.8%) of 43 patients receiving ruxolitinib and corticosteroids had a 50% or greater corticosteroid dose reduction from baseline. The most common treatment-emergent adverse events were anemia (64.8%), thrombocytopenia (62.0%), hypokalemia (49.3%), neutropenia (47.9%), and peripheral edema (45.1%). Ruxolitinib produced durable responses and encouraging survival compared with historical data in patients with steroid-refractory aGVHD who otherwise have dismal outcomes. The safety profile was consistent with expectations for ruxolitinib and this patient population.

Blinatumomab is currently approved for use as a single agent in relapsed and refractory acute lymphoblastic leukemia (ALL). Cytotoxicity is mediated via signaling through the T-cell receptor (TCR). There is now much interest in combining blinatumomab with targeted therapies, particularly in Philadelphia chromosome–positive ALL (Ph+ ALL). However, some second- and third-generation ABL inhibitors also potently inhibit Src family kinases that are important in TCR signaling. We combined ABL inhibitors and dual Src/ABL inhibitors with blinatumomab in vitro from both healthy donor samples and primary samples from patients with Ph+ ALL. Blinatumomab alone led to both T-cell proliferation and elimination of target CD19+ cells and enhanced production of interferon-γ (IFN-γ). The addition of the ABL inhibitors imatinib or nilotinib to blinatumomab did not inhibit T-cell proliferation or IFN-γ production. However, the addition of dasatinib or ponatinib inhibited T-cell proliferation and IFN-γ production. Importantly, there was no loss of CD19+ cells treated with blinatumomab plus dasatinib or ponatinib in healthy samples or samples with a resistant ABL T315I mutation by dasatinib in combination with blinatumomab. These in vitro findings bring pause to the excitement of combination therapies, highlighting the importance of maintaining T-cell function with targeted therapies.

Patients who develop steroid-refractory acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic cell transplantation have poor prognosis, highlighting an unmet therapeutic need. In this open-label phase 2 study (ClinicalTrials.gov identifier: NCT02953678), patients aged at least 12 years with grades II to IV steroid-refractory aGVHD were eligible to receive ruxolitinib orally, starting at 5 mg twice daily plus corticosteroids, until treatment failure, unacceptable toxicity, or death. The primary end point was overall response rate (ORR) at day 28; the key secondary end point was duration of response (DOR) at 6 months. As of 2 July 2018, 71 patients received at least 1 dose of ruxolitinib. Forty-eight of those patients (67.6%) had grade III/IV aGVHD at enrollment. At day 28, 39 patients (54.9%; 95% confidence interval, 42.7%-66.8%) had an overall response, including 19 (26.8%) with complete responses. Best ORR at any time was 73.2% (complete response, 56.3%). Responses were observed across skin (61.1%), upper (45.5%) and lower (46.0%) gastrointestinal tract, and liver (26.7%). Median DOR was 345 days. Overall survival estimate at 6 months was 51.0%. At day 28, 24 (55.8%) of 43 patients receiving ruxolitinib and corticosteroids had a 50% or greater corticosteroid dose reduction from baseline. The most common treatment-emergent adverse events were anemia (64.8%), thrombocytopenia (62.0%), hypokalemia (49.3%), neutropenia (47.9%), and peripheral edema (45.1%). Ruxolitinib produced durable responses and encouraging survival compared with historical data in patients with steroid-refractory aGVHD who otherwise have dismal outcomes. The safety profile was consistent with expectations for ruxolitinib and this patient population.

Highly active BTK inhibitors (BTKis) and the BCL2 inhibitor venetoclax have transformed the therapeutic landscape for chronic lymphocytic leukemia (CLL). Results of prospective clinical trials demonstrate the efficacy of venetoclax to salvage patients with disease progression on BTKis, but data on BTKi therapy after disease progression on venetoclax are limited, especially regarding durability of benefit. We retrospectively evaluated the records of 23 consecutive patients with relapsed/refractory CLL who received a BTKi (ibrutinib, n = 21; zanubrutinib, n = 2) after stopping venetoclax because of progressive disease. Median progression-free survival (PFS) and median overall survival after BTKi initiation were 34 months (range

Essential Thrombocythemia (ET) patients at high-risk of thrombosis require cytoreductive treatment, typically with hydroxycarbamide. Many patients are resistant or intolerant to hydroxycarbamide (HC-RES/INT) and are at increased risk of disease progression. MAJIC-ET is a randomized phase 2 study comparing ruxolitinib (RUX) to best available therapy (BAT) in HC-RES/INT ET, which showed no difference between the two arms in rates of hematological response or disease progression. The impact of additional non-MPN driver mutations (NDM) on the risk of disease complications in HC-RES/INT ET patients is unknown. Since the presence of NDM may influence trial outcomes, we expand the primary MAJIC-ET analysis to serially evaluate NDM in MAJIC-ET patients using a targeted myeloid 32-gene panel. NDM at baseline were detected in 30% of patients, most frequently affecting TET2 (11%) followed by TP53 (6.4%) and SF3B1 (6.4%). The presence of a NDM was associated with inferior 4-year transformation-free survival (TFS; 65.4% [95% CI 53.3 - 75%] vs. 82.8% [95% CI 73.2 - 89.1%], p=0.017). Specifically, TP53 (p=0.01) and splicing factor (SF, SF3B1, ZRSR2, SRSF2; p

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.

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.

The outcome of patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) is poor, particularly in patients ineligible for stem cell transplantation or who fail induction therapy or salvage therapy. The phase 1b portion of this open-label, dose-escalation (3+3+3 design) study examined the maximum tolerated dose (MTD) and preliminary safety and activity of the regimen in transplant-ineligible adults with histologically confirmed relapsed/refractory DLBCL after at least 1 prior therapy. Patients received once-daily 560 mg ibrutinib, 375 mg/m(2) intravenous rituximab day 1 of cycles 1 to 6, and 10, 15, 20, or 25 mg lenalidomide days 1 to 21 of each 28-day cycle. Forty-five patients were treated; median time since diagnosis was 14.1 months, and 51% of the patients had non–germinal center B-cell–like (non-GCB) DLBCL, 33% had transformed DLBCL, 60% were refractory, and 27% were primary refractory. Because of dose-limiting toxicities, a de-escalation cohort (10 mg lenalidomide) was initiated, and with subsequent re-escalation up to 25 mg lenalidomide, the MTD was not reached. In response-evaluable patients, the overall response rate (ORR) was 44% (complete response [CR], 28%); among them, the ORR was 65% (CR, 41%) in non-GCB and 69% and 56% in relapsed (n = 16) and secondary refractory (n = 27) disease, respectively. Overall and for non-GCB, median response duration was 15.9 months, with 2 patients receiving therapy beyond 3 years. Phase 2 was initiated with 20 mg lenalidomide in relapsed/refractory non-GCB, whereas the phase 1b 25-mg lenalidomide cohort was being completed; an additional 25-mg cohort in phase 2 is currently ongoing. This study was registered at www.clinicaltrials.gov as #NCT02077166.

Fostamatinib is a spleen tyrosine kinase inhibitor recently approved for the treatment of chronic immune thrombocytopenia (ITP) in patients without adequate response to at least 1 prior line of therapy. This article reviews fostmatinib’s mechanism of action and its clinical safety and efficacy in 2 industry-sponsored multicenter phase 3 randomized controlled trials in North America, Australia, and Europe (FIT1 and FIT2). Cost comparisons are discussed as well as the role of fostamatinib in relation to other options for chronic ITP.

The treatment of patients with Bing-Neel syndrome (BNS) is not standardized. We included patients with Waldenström macroglobulinemia (WM) and a radiologic and/or cytologic diagnosis of BNS treated with ibrutinib monotherapy. Response assessment was based on criteria for BNS from the 8th International Workshop for WM. Survival from BNS diagnosis (BNS survival), survival from ibrutinib initiation to last follow-up or death (ibrutinib survival), and time from ibrutinib initiation to ibrutinib discontinuation for toxicity, progression, or death (event-free survival [EFS]) were estimated. Twenty-eight patients were included in our study. The median age at BNS diagnosis was 65 years. Ibrutinib was the first line of treatment for BNS in 39% of patients. Ibrutinib was administered orally at a dose of 560 and 420 mg once daily in 46% and 54% of patients, respectively; symptomatic and radiologic improvements were seen in 85% and 60% of patients within 3 months of therapy. At best response, 85% of patients had improvement or resolution of BNS symptoms, 83% had improvement or resolution of radiologic abnormalities, and 47% had cleared the disease in the cerebrospinal fluid. The 2-year EFS rate with ibrutinib was 80% (95% confidence interval [CI], 58%-91%), the 2-year ibrutinib survival rate was 81% (95% CI, 49%-94%), and the 5-year BNS survival rate was 86% (95% CI, 63%-95%). Ibrutinib therapy is effective in patients with BNS and should be considered as a treatment option in these patients.

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.

Ibrutinib is a first-in-class inhibitor of Bruton tyrosine kinase (BTK) and has shown single-agent activity in recurrent/refractory central nervous system (CNS) lymphoma. Clinical responses are often transient or incomplete, suggesting a need for a combination therapy approach. We conducted a phase 1b clinical trial to explore the sequential combination of ibrutinib (560 or 840 mg daily dosing) with high-dose methotrexate (HD-MTX) and rituximab in patients with CNS lymphoma (CNSL). HD-MTX was given at 3.5 g/m2 every 2 weeks for a total of 8 doses (4 cycles; 1 cycle = 28 days). Ibrutinib was held on days of HD-MTX infusion and resumed 5 days after HD-MTX infusion or after HD-MTX clearance. Single-agent daily ibrutinib was administered continuously after completion of induction therapy until disease progression, intolerable toxicity, or death. We also explored next-generation sequencing of circulating tumor DNA (ctDNA) in cerebrospinal fluid (CSF) before and during treatment. The combination of ibrutinib, HD-MTX, and rituximab was tolerated with an acceptable safety profile (no grade 5 events, 3 grade 4 events). No dose-limiting toxicity was observed. Eleven of 15 patients proceeded to maintenance ibrutinib after completing 4 cycles of the ibrutinib/HD-MTX/rituximab combination. Clinical responses occurred in 12 of 15 patients (80%). Sustained tumor responses were associated with clearance of ctDNA from the CSF. This trial was registered at www.clinicaltrials.gov as #NCT02315326.

In the postrituximab era, approximately half of the patients with relapsed or refractory (rel/ref) diffuse large B-cell lymphoma (DLBCL) fail to achieve a chemosensitive response to standard salvage therapy, and are thus ineligible to proceed to autologous stem cell transplantation with curative intent. The Bruton tyrosine kinase inhibitor ibrutinib demonstrates single-agent activity in rel/ref DLBCL, particularly of non–germinal center (non-GC) cell of origin. We conducted a single-center phase 1 study evaluating dose-escalated ibrutinib, in a 3-by-3 design, in combination with rituximab, ifosfamide, carboplatin, and etoposide (R-ICE) in physiologically transplant-eligible rel/ref DLBCL patients. Twenty-one patients have been treated and are evaluable for toxicity with no dose-limiting toxicities observed through expansion with ibrutinib at 840 mg daily at dose level 3. Of the 20 patients evaluable for response, per modern International Conference on Malignant Lymphoma criteria, 11 patients achieved complete remission (CR) and 7 patients achieved partial remission for an overall response rate of 90%. All evaluable patients with non-GC DLBCL achieved a metabolic CR. Ibrutinib in combination with R-ICE demonstrates tolerability and efficacy in rel/ref DLBCL, particularly of non-GC phenotype. This treatment program warrants further investigation in later-phase studies. This trial was registered at www.clinicaltrials.gov as #NCT02219737.

Ruxolitinib (RUX)-based combinations may provide benefit for patients with myelofibrosis (MF). In this open-label, nonrandomized, prospective phase 2 study, patients with MF initially received RUX twice per day continuously in 28-day cycles for the first 3 cycles. Azacitidine (AZA) 25 mg/m2 (days 1-5) was added starting with cycle 4 and could be subsequently increased to 75 mg/m2 (days 1-5). Forty-six patients were enrolled with a median follow-up of 28 months (range, 4-50+ months). An International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) response was achieved in 33 patients (72%), with a median time to response of 1.8 months (range, 0.7-19.0 months). One-fourth (7 of 33) of the IWG-MRT responses occurred after the addition of AZA. A reduction of >50% in palpable spleen length at 24 weeks and at any time on the study was achieved in 62% and 71% of the evaluable patients, respectively. Among patients who achieved a >50% reduction in spleen length at 24 weeks, 95% had maintained it at 48 weeks. Notably, improvements in bone marrow reticulin fibrosis grade occurred in 57% of the patients at 24 months. Treatment discontinuations as a result of drug-related toxicities occurred in 4 patients (9%), all as a result of cytopenias. New onset grade 3 to 4 anemia and thrombocytopenia occurred in 35% and 26% of patients, respectively. RUX and AZA were safe, with encouraging spleen response rates and improvement in bone marrow fibrosis in patients with MF. This trial was registered at www.clinicaltrials.gov as #NCT01787487.

In this issue of Blood , Swords et al report on promising data from a phase 1b combination study of the hypomethylating agent 5-azacitidine and pevonedistat, an inhibitor of the NEDD8-activating enzyme. 1

To identify new therapeutic targets in acute myeloid leukemia (AML), we performed small-molecule and small-interfering RNA (siRNA) screens of primary AML patient samples. In 23% of samples, we found sensitivity to inhibition of colony-stimulating factor 1 (CSF1) receptor (CSF1R), a receptor tyrosine kinase responsible for survival, proliferation, and differentiation of myeloid-lineage cells. Sensitivity to CSF1R inhibitor GW-2580 was found preferentially in de novo and favorable-risk patients, and resistance to GW-2580 was associated with reduced overall survival. Using flow cytometry, we discovered that CSF1R is not expressed on the majority of leukemic blasts but instead on a subpopulation of supportive cells. Comparison of CSF1R-expressing cells in AML vs healthy donors by mass cytometry revealed expression of unique cell-surface markers. The quantity of CSF1R-expressing cells correlated with GW-2580 sensitivity. Exposure of primary AML patient samples to a panel of recombinant cytokines revealed that CSF1R inhibitor sensitivity correlated with a growth response to CSF1R ligand, CSF1, and other cytokines, including hepatocyte growth factor (HGF). The addition of CSF1 increased the secretion of HGF and other cytokines in conditioned media from AML patient samples, whereas adding GW-2580 reduced their secretion. In untreated cells, HGF levels correlated significantly with GW-2580 sensitivity. Finally, recombinant HGF and HS-5–conditioned media rescued cell viability after GW-2580 treatment in AML patient samples. Our results suggest that CSF1R-expressing cells support the bulk leukemia population through the secretion of HGF and other cytokines. This study identifies CSF1R as a novel therapeutic target of AML and provides a mechanism of paracrine cytokine/growth factor signaling in this 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.

Treatments for high-risk essential thrombocythemia (ET) address thrombocytosis, disease-related symptoms, as well as risks of thrombosis, hemorrhage, transformation to myelofibrosis, and leukemia. Patients resistant/intolerant to hydroxycarbamide (HC) have a poor outlook. MAJIC (ISRCTN61925716) is a randomized phase 2 trial of ruxolitinib (JAK1/2 inhibitor) vs best available therapy (BAT) in ET and polycythemia vera patients resistant or intolerant to HC. Here, findings of MAJIC-ET are reported, where the modified intention-to-treat population included 58 and 52 patients randomized to receive ruxolitinib or BAT, respectively. There was no evidence of improvement in complete response within 1 year reported in 27 (46.6%) patients treated with ruxolitinib vs 23 (44.2%) with BAT (P = .40). At 2 years, rates of thrombosis, hemorrhage, and transformation were not significantly different; however, some disease-related symptoms improved in patients receiving ruxolitinib relative to BAT. Molecular responses were uncommon; there were 2 complete molecular responses (CMR) and 1 partial molecular response in CALR-positive ruxolitinib-treated patients. Transformation to myelofibrosis occurred in 1 CMR patient, presumably because of the emergence of a different clone, raising questions about the relevance of CMR in ET patients. Grade 3 and 4 anemia occurred in 19% and 0% of ruxolitinib vs 0% (both grades) in the BAT arm, and grade 3 and 4 thrombocytopenia in 5.2% and 1.7% of ruxolitinib vs 0% (both grades) of BAT-treated patients. Rates of discontinuation or treatment switching did not differ between the 2 trial arms. The MAJIC-ET trial suggests that ruxolitinib is not superior to current second-line treatments for ET. This trial was registered at www.isrctn.com as #ISRCTN61925716.

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.

In the postrituximab era, approximately half of the patients with relapsed or refractory (rel/ref) diffuse large B-cell lymphoma (DLBCL) fail to achieve a chemosensitive response to standard salvage therapy, and are thus ineligible to proceed to autologous stem cell transplantation with curative intent. The Bruton tyrosine kinase inhibitor ibrutinib demonstrates single-agent activity in rel/ref DLBCL, particularly of non-germinal center (non-GC) cell of origin. We conducted a single-center phase 1 study evaluating dose-escalated ibrutinib, in a 3-by-3 design, in combination with rituximab, ifosfamide, carboplatin, and etoposide (R-ICE) in physiologically transplant-eligible rel/ref DLBCL patients. Twenty-one patients have been treated and are evaluable for toxicity with no dose-limiting toxicities observed through expansion with ibrutinib at 840 mg daily at dose level 3. Of the 20 patients evaluable for response, per modern International Conference on Malignant Lymphoma criteria, 11 patients achieved complete remission (CR) and 7 patients achieved partial remission for an overall response rate of 90%. All evaluable patients with non-GC DLBCL achieved a metabolic CR. Ibrutinib in combination with R-ICE demonstrates tolerability and efficacy in rel/ref DLBCL, particularly of non-GC phenotype. This treatment program warrants further investigation in later-phase studies. This trial was registered at www.clinicaltrials.gov as #NCT02219737.

To the editor: We have shown that it is feasible to administer a first-generation tyrosine kinase inhibitor (TKI) early after allogeneic hematopoietic cell transplantation (HCT) in patients with Philadelphia chromosome-positive (Ph+) leukemia.[1][1] The European Society for Blood and Marrow

Graft-versus-host disease (GVHD) is the major complication after allogeneic stem cell transplantation (SCT). Emerging evidence indicates that GVHD leads to injury of intestinal stem cells. However, it remains to be investigated whether skin stem cells could be targeted in skin GVHD. Lgr5+ hair follicle stem cells (HFSCs) contribute to folliculogenesis and have a multipotent capacity to regenerate all epithelial cells in repair. We studied the fate of Lgr5+ HFSCs after SCT and explored the novel treatment to protect Lgr5+ HFSCs against GVHD using murine models of SCT. We found that GVHD reduced Lgr5+ HFSCs in association with impaired hair regeneration and wound healing in the skin after SCT. Topical corticosteroids, a standard of care for a wide range of skin disorders including GVHD, damaged HFSCs and failed to improve skin homeostasis, despite of their anti-inflammatory effects. In contrast, JAK1/2 inhibitor ruxolitinib significantly ameliorated skin GVHD, protected Lgr5+ HFSCs, and restored hair regeneration and wound healing after SCT. We, for the first time, found that GVHD targets Lgr5+ HFSCs and that topical ruxolitinib represents a novel strategy to protect skin stem cells and maintain skin homeostasis in GVHD.

Despite significant improvements in the signs and symptoms of myelofibrosis (MF), and possible prolongation of patients' survival, some have disease that is refractory to ruxolitinib and many lose their response over time. Furthermore, patients with ≥3 mutations are less likely to respond to ruxolitinib. Here we describe outcomes after ruxolitinib discontinuation in MF patients enrolled in a phase 1/2 study at our center. After a median follow-up of 79 months, 86 patients had discontinued ruxolitinib (30 of whom died while on therapy). The median follow-up after ruxolitinib discontinuation for the remaining 56 patients was 32 months, with median survival after discontinuation of 14 months. Platelets

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.

Acute and chronic leukemias, including CD34(+) CML cells, demonstrate increased expression of the Wilms tumor gene 1 product (WT1), making WT1 an attractive therapeutic target. However, WT1 is a currently undruggable, intracellular protein. ESKM is a human IgG1 T-cell receptor mimic monoclonal antibody directed to a 9-amino acid sequence of WT1 in the context of cell surface HLA-A*02. ESKM was therapeutically effective, alone and in combination with tyrosine kinase inhibitors (TKIs), against Philadelphia chromosome-positive acute leukemia in murine models, including a leukemia with the most common, pan-TKI, gatekeeper resistance mutation, T315I. ESKM was superior to the first-generation TKI, imatinib. Combination therapy with ESKM and TKIs was superior to either drug alone, capable of curing mice. ESKM showed no toxicity to human HLA-A*02:01(+) stem cells under the conditions of this murine model. These features of ESKM make it a promising nontoxic therapeutic agent for sensitive and resistant Ph(+) leukemias.

We aimed to evaluate the impact of pretransplant imatinib administration on the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in adults with Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL). We retrospectively analyzed 738 patients with Ph(+) ALL that underwent allo-HSCT between 1990 and 2010 using data from the Transplant Registry Unified Management Program of the Japan Society of Hematopoietic Cell Transplantation. We compared the allo-HSCT outcomes between 542 patients who received imatinib before allo-HSCT during the initial complete remission period (imatinib cohort) and 196 patients who did not receive imatinib (non-imatinib cohort). The 5-year overall survival after allo-HSCT was significantly higher in the imatinib cohort than in the non-imatinib cohort (59% vs 38%; 95% confidence interval [CI], 31-45%; P < .001). Multivariate analysis indicated that pretransplant imatinib administration had beneficial effects on overall survival (hazard ratio [HR], 0.57; 95% CI, 0.42-0.77; P < .001), relapse (HR, 0.66; 95% CI, 0.43-0.99; P = .048), and nonrelapse mortality (HR, 0.55; 95% CI, 0.37-0.83; P = .005). In conclusion, our study showed that imatinib administration before allo-HSCT had advantageous effects on the clinical outcomes of allo-HSCT in patients with Ph(+) ALL.