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Abstract Clinical bleeding events are reported here from 773 patients with B‐cell malignancies receiving pirtobrutinib monotherapy from the phase 1/2 BRUIN study (ClinicalTrials.gov identifier: NCT03740529), either in the presence or absence of antithrombotic therapy (antithrombotic exposed [AT‐E], n = 216; antithrombotic nonexposed [AT‐NE], n = 557). Among the AT‐E cohort, 51.9% received platelet aggregation inhibitors, 36.6% received direct factor Xa inhibitors, 18.5% received heparins, 5.6% received salicylic acid for indications other than platelet aggregation inhibition, and 2.3% received thrombolytics. Warfarin was not permitted. Any‐grade bleeding/bruising events occurred in 97 patients (44.9%; 95% confidence interval [CI], 38.3–51.5) in the AT‐E cohort and 181 patients (32.5%; 95% CI, 28.6–36.4) in the AT‐NE cohort. Most bleeding/bruising events in both cohorts began within the first 6 months of treatment (AT‐E: 65.4%; AT‐NE: 72.5%). Contusion was the most common bleeding/bruising event in both cohorts (AT‐E: 22.7%; AT‐NE: 18.1%). Grade ≥3 bleeding/bruising events were reported in six patients (2.8%) in the AT‐E cohort and 11 patients (2.0%) in the AT‐NE cohort. Bleeding/bruising events requiring or prolonging hospitalization were reported in 2.3% and 1.6% of patients in the AT‐E and AT‐NE cohorts, respectively. No bleeding/bruising events led to pirtobrutinib dose reduction or permanent discontinuation in the AT‐E cohort, and one patient (0.2%) in the AT‐NE cohort experienced an event requiring dose reduction. These data support the safety of pirtobrutinib in patients requiring antithrombotic therapies.

Bruton tyrosine kinase inhibitors (BTKi) have transformed the treatment of B-cell malignancies, but intolerance has often led to their discontinuation. The phase 1/2 BRUIN study evaluated pirtobrutinib, a highly selective non-covalent (reversible) BTKi, in patients with R/R B-cell malignancies (NCT03740529). Pirtobrutinib was investigated in 127 patients with intolerance to at least one prior BTKi therapy in the absence of progressive disease. The most common adverse event (AE) leading to BTKi discontinuation was cardiac disorders (n=40, 31.5%), specifically atrial fibrillation (n=30, 23.6%). The median follow-up was 17.4 months and the median time on pirtobrutinib was 15.3 months. The most common reasons for pirtobrutinib discontinuation were progressive disease (26.8%), AE (10.2%), or death (5.5%). The most frequent treatment-emergent AEs were fatigue (39.4%) and neutropenia (37.0%). Among patients who discontinued a prior BTKi for a cardiac issue, 75% had no recurrence of their cardiac AE. No patient discontinued pirtobrutinib for the same AE that led to discontinuation of the prior BTKi. In 78 CLL/SLL and 21 MCL patients intolerant to prior BTKi, ORR to pirtobrutinib was 76.9% and 81.0%, respectively. Median PFS for CLL/SLL was 28.4 months and was not estimable for MCL. These results suggest that pirtobrutinib was safe, well-tolerated, and an efficacious option in patients with prior BTKi-intolerance.

Abstract Acute myeloid leukemia (AML) is a highly aggressive hematologic cancer with poor survival across a broad range of molecular subtypes. Development of efficacious and well-tolerable therapies encompassing the range of mutations that can arise in AML remains an unmet need. The bromo- and extra-terminal domain (BET) family of proteins represents an attractive therapeutic target in AML due to their crucial roles in many cellular functions, regardless of any specific mutation. Many BET inhibitors (BETi) are currently in pre-clinical and early clinical development, but acquisition of resistance continues to remain an obstacle for the drug class. Novel methods to circumvent this development of resistance could be instrumental for the future use of BET inhibitors in AML, both as monotherapy and in combination. To date, many investigations into possible drug combinations of BETi with CDK inhibitors have focused on CDK9, which has a known physical and functional interaction with the BET protein BRD4. Therefore, we wished to investigate possible synergy and additive effects between inhibitors of these targets in AML. Here, we describe combination therapy with the multi-CDK inhibitor dinaciclib and the BETi PLX51107 in pre-clinical models of AML. Dinaciclib and PLX51107 demonstrate additive effects in AML cell lines, primary AML samples, and in vivo. Further, we demonstrate novel activity of dinaciclib through inhibition of the canonical/β-catenin dependent Wnt signaling pathway, a known resistance mechanism to BETi in AML. We show dinaciclib inhibits Wnt signaling at multiple levels, including downregulation of β-catenin, the Wnt co-receptor LRP6, as well as many Wnt pathway components and targets. Moreover, dinaciclib sensitivity remains unaffected in a setting of BET resistance, demonstrating similar inhibitory effects on Wnt signaling when compared to BET-sensitive cells. Ultimately, our results demonstrate rationale for combination CDKi and BETi in AML. In addition, our novel finding of Wnt signaling inhibition could have potential implications in other cancers where Wnt signaling is dysregulated and demonstrates one possible approach to circumvent development of BET resistance in AML.

Patients with chronic lymphocytic leukemia (CLL) respond well to initial treatment with the Bcell lymphoma 2 (BCL2) inhibitor venetoclax. Upon relapse, they often retain sensitivity to BCL2 targeting, but durability of response remains a concern. We hypothesize that targeting both BCL2 and B-cell lymphoma-extra large (BCLXL) will be a successful strategy to treat CLL, including for patients who relapse on venetoclax. To test this hypothesis, we conducted a pre-clinical investigation of LP-118, a highly potent inhibitor of BCL2 with moderate BCLXL inhibition to minimize platelet toxicity. This study demonstrated that LP-118 induces efficient BAK activation, cytochrome C release, and apoptosis in both venetoclax naïve and resistant CLL cells. Significantly, LP-118 is effective in cell lines expressing the BCL2 G101V mutation and in cells expressing BCLXL but lacking BCL2 dependence. Using an immunocompetent mouse model, Eμ-TCL1, LP-118 demonstrates low platelet toxicity, which hampered earlier BCLXL inhibitors. Finally, LP-118 in the RS4;11 and OSU-CLL xenograft models results in decreases in tumor burden and survival advantage, respectively. These results provide a mechanistic rationale for the evaluation of LP-118 for the treatment of venetoclax responsive and relapsed CLL.

IntroductionTherapeutic antibodies have become a major strategy to treat oncologic diseases. For chronic lymphocytic leukemia, antibodies against CD20 are used to target and elicit cytotoxic responses against malignant B cells. However, efficacy is often compromised due to a suppressive microenvironment that interferes with cellular immune responses. To overcome this suppression, agonists of pattern recognition receptors have been studied which promote direct cytotoxicity or elicit anti-tumoral immune responses. NOD2 is an intracellular pattern recognition receptor that participates in the detection of peptidoglycan, a key component of bacterial cell walls. This detection then mediates the activation of multiple signaling pathways in myeloid cells. Although several NOD2 agonists are being used worldwide, the potential benefit of these agents in the context of antibody therapy has not been explored.MethodsPrimary cells from healthy-donor volunteers (PBMCs, monocytes) or CLL patients (monocytes) were treated with versus without the NOD2 agonist L18-MDP, then antibody-mediated responses were assessed. In vivo, the Eµ-TCL1 mouse model of CLL was used to test the effects of L18-MDP treatment alone and in combination with anti-CD20 antibody.ResultsTreatment of peripheral blood mononuclear cells with L18-MDP led to activation of monocytes from both healthy donors and CLL patients. In addition, there was an upregulation of activating FcγR in monocytes and a subsequent increase in antibody-mediated phagocytosis. This effect required the NF-κB and p38 signaling pathways. Treatment with L18-MDP plus anti-CD20 antibody in the Eµ-TCL model of CLL led to a significant reduction of CLL load, as well as to phenotypic changes in splenic monocytes and macrophages.ConclusionsTaken together, these results suggest that NOD2 agonists help overturn the suppression of myeloid cells, and may improve the efficacy of antibody therapy for CLL.

Abstract Inhibitors of B cell receptor (BCR) signaling such as the Bruton’s tyrosine kinase (BTK) inhibitors are effective therapeutics for chronic lymphocytic leukemia (CLL). The first-in-class covalent BTK inhibitor, ibrutinib, produces durable responses in most CLL patients; however, complete responses are only observed in a minority of patients. B cell lymphoma 2 (BCL2), an anti-apoptotic protein that contributes to CLL cell survival, has also been investigated as a therapeutic target. The BCL2 inhibitor venetoclax is effective in patients with CLL and can produce undetectable minimal residual disease, allowing discontinuation of therapy. In combination, ibrutinib and venetoclax have shown preclinical synergy and clinical efficacy. Nemtabrutinib is a next generation, reversible inhibitor of BTK that potently inhibits BCR signaling in treatment-naïve and ibrutinib-refractory CLL cells ex vivo. The clinical efficacy of combining BTK inhibitors with BCL2 inhibitors motivated us to evaluate the novel combination of nemtabrutinib and venetoclax. In vitro studies show that nemtabrutinib and venetoclax are not antagonistic to each other. In an adoptive transfer CLL mouse model, mice treated with nemtabrutinib and venetoclax had prolonged survival compared to mice treated with ibrutinib and venetoclax. Our preclinical studies further validate the combination of BTK inhibitors with venetoclax and justify further investigation of combining nemtabrutinib with venetoclax in CLL.

We assessed vaccine-induced antibody responses to the SARS-CoV-2 ancestral virus and Omicron variant before and after booster immunization in 57 patients with B cell malignancies. Over one-third of vaccinated patients at the pre-booster time point were seronegative, and these patients were predominantly on active cancer therapies such as anti-CD20 monoclonal antibody. While booster immunization was able to induce detectable antibodies in a small fraction of seronegative patients, the overall booster benefit was disproportionately evident in patients already seropositive and not receiving active therapy. While ancestral virus– and Omicron variant–reactive antibody levels among individual patients were largely concordant, neutralizing antibodies against Omicron tended to be reduced. Interestingly, in all patients, including those unable to generate detectable antibodies against SARS-CoV-2 spike, we observed comparable levels of EBV- and influenza-reactive antibodies, demonstrating that B cell–targeting therapies primarily impair de novo but not preexisting antibody levels. These findings support rationale for vaccination before cancer treatment.

Some genes that are part of balanced translocations are reported as drivers for tumourigenesis. Here, the authors report a translocation involving MTCP1 in chronic lymphocytic leukemia and show that MTCP1 overexpression leads to the disease in a murine model.

Abstract B-cell receptor (BCR) antagonists such as the BTK inhibitor ibrutinib have proven to effectively target chronic lymphocytic leukemia (CLL) tumor cells, leading to impressive response rates in these patients. However patients do still relapse on ibrutinib, and the progressive disease is often quite aggressive requiring immediate treatment. Several strategies are being pursued to treat patients who relapse on ibrutinib therapy. As the most common form of relapse is the development of a mutant form of BTK which limits ibrutinib binding, agents which lead to degradation of the BTK protein are a promising strategy. Our study explores the efficacy of the Hsp90 inhibitor, SNX-5422, in CLL. The SNX Hsp90 inhibitor was effective in primary CLL cells, as well as B-cell lines expressing either BTK wild type or C481 mutant BTK, which has been identified as the primary resistance mechanism to ibrutinib in CLL patients. Furthermore the combination of SNX-5422 and ibrutinib provided a remarkable in vivo survival benefit in the Eμ-TCL1 mouse model of CLL compared to the vehicle or single agent groups (51 day median survival in the vehicle and ibrutinib groups versus 100 day median survival in the combination). We report here preclinical data suggesting that the Hsp90 inhibitor SNX-5422, which has been pursued in clinical trials in both solid tumor and hematological malignancies, is a potential therapy for ibrutinib resistant CLL.

Abstract Background Exportin 1 (XPO1/CRM1) is a key mediator of nuclear export with relevance to multiple cancers, including chronic lymphocytic leukemia (CLL). Whole exome sequencing has identified hot-spot somatic XPO1 point mutations which we found to disrupt highly conserved biophysical interactions in the NES-binding groove, conferring novel cargo-binding abilities and forcing cellular mis-localization of critical regulators. However, the pathogenic role played by change-in-function XPO1 mutations in CLL is not fully understood. Methods We performed a large, multi-center retrospective analysis of CLL cases (N = 1286) to correlate nonsynonymous mutations in XPO1 (predominantly E571K or E571G; n = 72) with genetic and epigenetic features contributing to the overall outcomes in these patients. We then established a mouse model with over-expression of wildtype (wt) or mutant (E571K or E571G) XPO1 restricted to the B cell compartment (Eµ-XPO1). Eµ-XPO1 mice were then crossed with the Eµ-TCL1 CLL mouse model. Lastly, we determined crystal structures of XPO1 (wt or E571K) bound to several selective inhibitors of nuclear export (SINE) molecules (KPT-185, KPT-330/Selinexor, and KPT-8602/Eltanexor). Results We report that nonsynonymous mutations in XPO1 associate with high risk genetic and epigenetic features and accelerated CLL progression. Using the newly-generated Eµ-XPO1 mouse model, we found that constitutive B-cell over-expression of wt or mutant XPO1 could affect development of a CLL-like disease in aged mice. Furthermore, concurrent B-cell expression of XPO1 with E571K or E571G mutations and TCL1 accelerated the rate of leukemogenesis relative to that of Eµ-TCL1 mice. Lastly, crystal structures of E571 or E571K-XPO1 bound to SINEs, including Selinexor, are highly similar, suggesting that the activity of this class of compounds will not be affected by XPO1 mutations at E571 in patients with CLL. Conclusions These findings indicate that mutations in XPO1 at E571 can drive leukemogenesis by priming the pre-neoplastic lymphocytes for acquisition of additional genetic and epigenetic abnormalities that collectively result in neoplastic transformation.

PROTACs have emerged as promising therapeutic agents but their cellular uptake is often inefficient. Here, the authors show that reversible covalent warhead chemistry improves PROTAC intracellular accumulation and target engagement, and develop a dual inhibitor/degrader of Bruton’s tyrosine kinase

In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) are a common mechanism by which intercellular messages are communicated between normal, diseased, and transformed cells. Studies of EVs in CLL and other cancers have great variability and often lack reproducibility. For CLL patient plasma and cell lines, we sought to characterize current approaches used in isolating EV products and understand whether cell culture–conditioned media or complex biological fluids confound results. Utilizing nanoparticle tracking analysis, protein quantification, and electron microscopy, we show that ultracentrifugation with an OptiPrep cushion can effectively minimize contaminants from starting materials including plasma and conditioned media of CLL cell lines grown in EV-depleted complete RPMI media but not grown in the serum-free media AIM V commonly used in CLL experimental work. Moreover, we confirm the benefit of including 25 mM trehalose in PBS during EV isolation steps to reduce EV aggregation, to preserve function for downstream applications and characterization. Furthermore, we report the highest particles/μg EVs were obtained from our CLL cell lines utilizing the CELLine bioreactor flask. Finally, we optimized a proliferation assay that offers a functional evaluation of our EVs with minimal sample requirements.

Older patients account for the majority of patients with chronic lymphocytic leukemia (CLL), and so strategies for managing CLL in this population is of upmost importance. Inhibition of Bruton’s tyrosine kinase (BTK) has been a successful therapeutic strategy in CLL, and the first-generation BTK inhibitor ibrutinib has been shown to be superior to standard chemoimmunotherapy in multiple studies specifically targeting older patients. A second-generation BTK inhibitor, acalabrutinib, has also been studied in CLL, and has recently been granted breakthrough designation by the United States Food and Drug Administration. One ongoing question is whether the addition of anti-CD20 monoclonal antibodies improve response or response durability with BTK inhibitors. In this review, we will discuss clinical trials of ibrutinib and acalabrutinib in older patients with CLL, and the possible contributions of anti-CD20 antibodies to these therapies.

Abstract Evaluating population‐based data of hematologic malignancies (HMs) in older adults provides prognostic information for this growing demographic. Incidence rates and one‐ and five‐year relative survival rates were examined for specific HMs among adults ages ≥75 years using data from the Surveillance, Epidemiology and End Results (SEER) Program. Hematologic malignancy cases (Hodgkin lymphoma (HL), non‐Hodgkin lymphoma (NHL), multiple myeloma (MM), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML)) were reported to one of 18 SEER registries. Recent average annual (2010–2014) incidence rates and incidence trends from 1973 to 2014 were examined for cases ages ≥75 years. One‐ and five‐year relative cancer survival rates were examined for adults ages ≥75 years diagnosed 2007–2013, with follow‐up into 2014. From 1973 to 2014, incidence rates increased for NHL, MM, and AML, decreased for HL, and remained relatively stable for ALL, CLL, and CML among adults ages ≥75 years. The highest one‐ and five‐year relative survival rates were observed among adults with CLL ages 75–84 years (1 year: 91.8% (95% CI = 91.8–90.8)) and 5 years: 76.5% (95% CI = 74.2–78.6)). The lowest one‐ and five‐year survival rates were observed among adults with AML ages 75–84 (1 year: 18.2% (95% CI = 74.2–78.6) and 5 years: 2.7% (95% CI = 2.0–3.6)). Survival for older adults ages ≥75 years with HMs is poor, particularly for acute leukemia. Understanding the heterogeneity in HM outcomes among older patients may help clinicians better address the hematological cancer burden and mortality in the aging population.

Abstract: Ibrutinib is a highly effective targeted therapy for chronic lymphocytic leukemia (CLL). However, ibrutinib must be discontinued in a subset of patients due to progressive CLL or transformation to aggressive lymphoma (Richter transformation). Transformation occurs early in the course of therapy and has an extremely poor prognosis. Thus, identification of prognostic markers associated with transformation is of utmost importance. Near-tetraploidy (4 copies of most chromosomes within a cell) has been reported in various lymphomas, but its incidence and significance in CLL has not been described. Using fluorescence in situ hybridization, we detected near-tetraploidy in 9 of 297 patients with CLL prior to beginning ibrutinib treatment on 1 of 4 clinical trials (3.0%; 95% confidence interval [CI], 1.4%-5.7%). Near-tetraploidy was associated with aggressive disease characteristics: Rai stage 3/4 (P = .03), deletion 17p (P = .03), and complex karyotype (P = .01). Near-tetraploidy was also associated with ibrutinib discontinuation due to Richter transformation (P < .0001), but not due to progressive CLL (P = .41). Of the 9 patients with near-tetraploidy, 6 had Richter transformation with diffuse large B-cell lymphoma. In a multivariable model, near-tetraploidy (hazard ratio [HR], 8.66; 95% CI, 3.83-19.59; P < .0001) and complex karyotype (HR, 4.77; 95% CI, 1.42-15.94; P = .01) were independent risk factors for discontinuing ibrutinib due to transformation. Our results suggest that near-tetraploidy is a potential prognostic marker for Richter transformation to assess in patients going on ibrutinib.

Abstract The introduction of miR profiling of chronic lymphocytic leukemia (CLL) patients with different cytogenetic profiles and responses to therapy has allowed incorporation of important miR‐mRNA interactions into the understanding of disease biology. In this study, we performed miR expression analysis using NanoString nCounter to discover differentially regulated miRs after therapy with the Bruton tyrosine kinase inhibitor ibrutinib. Of the differentially regulated miRs in the discovery set, miR‐29c and miR‐126 were confirmed using real‐time PCR to be upregulated in CLL patient cells with ibrutinib therapy. In the validation set, an inverse correlation was observed between miR‐126 levels and expression of its putative target p85β, an isoform of the phosphoinositide 3‐kinase p85 regulatory subunit. We found that mRNA for the host gene EGFL7, primary unprocessed miR‐126, and mature miR‐126 are all downregulated in CLL cells compared to normal B cells. Patients in later stages of disease have a greater decrease in miR‐126 expression compared to treatment‐naive patients, indicating that lower miR‐126 levels may associate with disease progression. Overexpression of miR‐126 in leukemia cell lines significantly downregulates p85β expression and decreases activation of prosurvival mitogen‐activated protein kinase (MAPK) signaling. These results implicate miR‐126 in the pathology of CLL.