Modulation of RNA splicing enhances response to BCL2 inhibition in leukemia.

Therapy resistance is a major challenge in the treatment of cancer. Here, we performed CRISPR-Cas9 screens across a broad range of therapies used in acute myeloid leukemia to identify genomic determinants of drug response. Our screens uncover a selective dependency on RNA splicing factors whose loss preferentially enhances response to the BCL2 inhibitor venetoclax. Loss of the splicing factor RBM10 augments response to venetoclax in leukemia yet is completely dispensable for normal hematopoiesis. Combined RBM10 and BCL2 inhibition leads to mis-splicing and inactivation of the inhibitor of apoptosis XIAP and downregulation of BCL2A1, an anti-apoptotic protein implicated in venetoclax resistance. Inhibition of splicing kinase families CLKs (CDC-like kinases) and DYRKs (dual-specificity tyrosine-regulated kinases) leads to aberrant splicing of key splicing and apoptotic factors that synergize with venetoclax, and overcomes resistance to BCL2 inhibition. Our findings underscore the importance of splicing in modulating response to therapies and provide a strategy to improve venetoclax-based treatments

Kinase-Impaired BTK Mutations Are Susceptible to Clinical-Stage BTK and IKZF1/3 Degrader NX-2127

INTRODUCTION: Bruton’s tyrosine kinase (BTK) is a nonreceptor kinase in the B cell receptor (BCR) signaling cascade critical for B cell survival. As such, chronic lymphocytic leukemia (CLL) and other B cell cancers are sensitive to inhibition of BTK. Covalent and noncovalent inhibitors of BTK have revolutionized the treatment of these cancers. Therefore, understanding mechanisms by which acquired mutation in BTK confer drug resistance and developing new therapies to overcome resistance are critically important. RATIONALE: We recently discovered BTK mutations that confer resistance across covalent and noncovalent BTK inhibitors. In this study, we found that a group of these mutants impair BTK kinase activity despite still enabling downstream BCR signaling. We therefore set out to understand the nonenzymatic functions of BTK and explored targeted protein degradation to overcome the oncogenic scaffold function of mutant BTK. This effort included evaluation of BTK degradation in patients with CLL treated in a phase 1 clinical trial of NX-2127, a first-in-class BTK degrader (NCT04830137). RESULTS: BTK enzymatic activity assays revealed that drug resistance mutations in BTK fall into two distinct groups: kinase proficient and kinase impaired. Immunoprecipitation mass spectrometry of kinase-impaired BTK L528W (Leu528→Trp) revealed a scaffold function of BTK with downstream signaling and survival dependent on surrogate kinases that bind to kinase-impaired BTK proteoforms. To target the nonenzymatic functions of BTK, we developed NX-2127, a heterobifunctional molecule that engages the ubiquitin-proteasome system to simultaneously bind both BTK and the cereblon E3 ubiquitin ligase complex, inducing polyubiquitination and proteasome-dependent degradation of IKZF1/3 and all recurrent drug-resistant forms of mutant BTK. The activity of NX-2127 on BTK degradation was further demonstrated in patients with CLL treated in a phase 1 clinical trial of NX-2127, where \u3e80% BTK degradation was achieved and clinical responses were also seen in 79% of evaluable patients, independent of mutant BTK genotypes. CONCLUSION: We identified that BTK inhibitor resistance mutations fall into two distinct functional categories. Kinase-impaired BTK mutants disable BTK kinase activity while promoting physical interactions with other kinases to sustain downstream BCR signaling. This scaffold function of BTK was disrupted by NX-2127, a potent BTK degrader, which showed promising responses for patients with relapsed and refractory CLL, independently of mutant BTK functional category

Mechanisms of Resistance to Noncovalent Bruton's Tyrosine Kinase Inhibitors

BackgroundCovalent (irreversible) Bruton's tyrosine kinase (BTK) inhibitors have transformed the treatment of multiple B-cell cancers, especially chronic lymphocytic leukemia (CLL). However, resistance can arise through multiple mechanisms, including acquired mutations in BTK at residue C481, the binding site of covalent BTK inhibitors. Noncovalent (reversible) BTK inhibitors overcome this mechanism and other sources of resistance, but the mechanisms of resistance to these therapies are currently not well understood.MethodsWe performed genomic analyses of pretreatment specimens as well as specimens obtained at the time of disease progression from patients with CLL who had been treated with the noncovalent BTK inhibitor pirtobrutinib. Structural modeling, BTK-binding assays, and cell-based assays were conducted to study mutations that confer resistance to noncovalent BTK inhibitors.ResultsAmong 55 treated patients, we identified 9 patients with relapsed or refractory CLL and acquired mechanisms of genetic resistance to pirtobrutinib. We found mutations (V416L, A428D, M437R, T474I, and L528W) that were clustered in the kinase domain of BTK and that conferred resistance to both noncovalent BTK inhibitors and certain covalent BTK inhibitors. Mutations in BTK or phospholipase C gamma 2 (PLCγ2), a signaling molecule and downstream substrate of BTK, were found in all 9 patients. Transcriptional activation reflecting B-cell-receptor signaling persisted despite continued therapy with noncovalent BTK inhibitors.ConclusionsResistance to noncovalent BTK inhibitors arose through on-target BTK mutations and downstream PLCγ2 mutations that allowed escape from BTK inhibition. A proportion of these mutations also conferred resistance across clinically approved covalent BTK inhibitors. These data suggested new mechanisms of genomic escape from established covalent and novel noncovalent BTK inhibitors. (Funded by the American Society of Hematology and others.)

BH3-mimetic toolkit guides the respective use of BCL2 and MCL1 BH3-mimetics in myeloma treatment: Disease progression favors MCL1 priming in myeloma

International audienceBH3-mimetics are promising drugs for hematologic malignancies that trigger cell death by promoting the release of pro-apoptotic BCL2 family members from anti-apoptotic proteins. Multiple myeloma is considered to be a disease dependent mainly on MCL1 for survival based mostly on studies using cell lines. We used a BH3-mimetic toolkit to study the dependency on BCL2, BCLXL or MCL1 in malignant plasma cells from 60 patients. Dependencies were analyzed using an unbiased BH3-mimetics cell-death clustering by k-means. In the whole cohort of patients, BCL2 dependency was mostly found in the CCND1 subgroup (83%). Of note, MCL1 dependence significantly increased from 33% at diagnosis to 69% at relapse, suggesting a plasticity of the cellular dependency favoring MCL1 dependencies at relapse. In addition, 35% of overall patient samples showed co-dependencies on either BCL2/MCL1 or BCLXL/MCL1. Finally, we identified a group of patients not targeted by any of the BH3-mimetics, predominantly at diagnosis in patients not presenting the common recurrent translocations. Mechanistically, we demonstrated that BAK is crucial for cell death induced by MCL1 mimetic A1210477, according to the protection from cell death observed by BAK knock-down as well as the complete and early disruption of MCL1/BAK complexes upon A1210477 treatment. Interestingly, this complex was also dissociated in A1210477 resistant cells, but free BAK was simultaneously recaptured by BCLXL, supporting the role of BCLXL in A1210477 resistance. In conclusion, our study opens the way to rationally use venetoclax and/or MCL1 BH3-mimetics for clinical evaluation in myeloma both at diagnosis and relapse

Clinical characteristics and outcomes of relapsed follicular lymphoma after autologous stem cell transplantation in the rituximab era

International audienceHigh-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is a therapeutic option for patients with relapsed follicular lymphoma (FL). The clinical characteristics and outcomes of FL relapse after ASCT in the rituximab era have not yet been fully elucidated. We retrospectively reviewed 414 FL patients treated with ASCT between 2000 and 2014 in four hematology departments. All patients received rituximab as a first-line treatment. We specifically analyzed the clinical characteristics, treatment strategies at relapse, and outcomes of 95 patients (23%) who relapsed after ASCT. The patients (median age, 57 y) received a median of two lines of therapy (range, 2-6) prior to ASCT, with 92% in complete response (CR) or partial response (PR) before ASCT. Histological transformation at relapse after ASCT was observed in 20% of the patients. Treatment at relapse after ASCT consisted of chemotherapy with or without rituximab (n = 45/90, 50%), targeted agents (18%), rituximab monotherapy (14%), or consolidation allogeneic transplantation after induction chemotherapy (12%) and radiotherapy (6%). After relapse, the median progression-free survival (PFS) and overall survival (OS) were 1 year (95% CI, 0.541-1.579) and 5.5 years (95% CI, 1.910-9.099), respectively. In the multivariate analysis, histological transformation (HT) was associated with OS (P = .044; HR 2.439; 95% CI, 1.025-5.806), and a high FLIPI score at relapse was associated with PFS (P = .028; HR 2.469; 95% CI, 1.104-5.521). This retrospective study showed that the period of PFS of patients who relapsed after ASCT is short. A biopsy should be performed for these patients to document the HT. Our results indicate that new treatment strategies will need to be developed for these patients

Kinase-impaired BTK mutations are susceptible to clinical-stage BTK and IKZF1/3 degrader NX-2127

Increasing use of covalent and noncovalent inhibitors of Bruton's tyrosine kinase (BTK) has elucidated a series of acquired drug-resistant BTK mutations in patients with B cell malignancies. Here we identify inhibitor resistance mutations in BTK with distinct enzymatic activities, including some that impair BTK enzymatic activity while imparting novel protein-protein interactions that sustain B cell receptor (BCR) signaling. Furthermore, we describe a clinical-stage BTK and IKZF1/3 degrader, NX-2127, that can bind and proteasomally degrade each mutant BTK proteoform, resulting in potent blockade of BCR signaling. Treatment of chronic lymphocytic leukemia with NX-2127 achieves >80% degradation of BTK in patients and demonstrates proof-of-concept therapeutic benefit. These data reveal an oncogenic scaffold function of mutant BTK that confers resistance across clinically approved BTK inhibitors but is overcome by BTK degradation in patients