Centre for Cancer Research - Research Publications
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ItemNo Preview AvailableEnhancer retargeting of CDX2 and UBTF::ATXN7L3 define a subtype of high-risk B-progenitor acute lymphoblastic leukemia(AMER SOC HEMATOLOGY, 2022-06-16)Transcriptome sequencing has identified multiple subtypes of B-progenitor acute lymphoblastic leukemia (B-ALL) of prognostic significance, but a minority of cases lack a known genetic driver. Here, we used integrated whole-genome (WGS) and -transcriptome sequencing (RNA-seq), enhancer mapping, and chromatin topology analysis to identify previously unrecognized genomic drivers in B-ALL. Newly diagnosed (n = 3221) and relapsed (n = 177) B-ALL cases with tumor RNA-seq were studied. WGS was performed to detect mutations, structural variants, and copy number alterations. Integrated analysis of histone 3 lysine 27 acetylation and chromatin looping was performed using HiChIP. We identified a subset of 17 newly diagnosed and 5 relapsed B-ALL cases with a distinct gene expression profile and 2 universal and unique genomic alterations resulting from aberrant recombination-activating gene activation: a focal deletion downstream of PAN3 at 13q12.2 resulting in CDX2 deregulation by the PAN3 enhancer and a focal deletion of exons 18-21 of UBTF at 17q21.31 resulting in a chimeric fusion, UBTF::ATXN7L3. A subset of cases also had rearrangement and increased expression of the PAX5 gene, which is otherwise uncommon in B-ALL. Patients were more commonly female and young adult with median age 35 (range,12-70 years). The immunophenotype was characterized by CD10 negativity and immunoglobulin M positivity. Among 16 patients with known clinical response, 9 (56.3%) had high-risk features including relapse (n = 4) or minimal residual disease >1% at the end of remission induction (n = 5). CDX2-deregulated, UBTF::ATXN7L3 rearranged (CDX2/UBTF) B-ALL is a high-risk subtype of leukemia in young adults for which novel therapeutic approaches are required.
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ItemNo Preview AvailableSingle-cell multiomics reveal the scale of multilayered adaptations enabling CLL relapse during venetoclax therapy(AMER SOC HEMATOLOGY, 2022-11-17)Venetoclax (VEN) inhibits the prosurvival protein BCL2 to induce apoptosis and is a standard therapy for chronic lymphocytic leukemia (CLL), delivering high complete remission rates and prolonged progression-free survival in relapsed CLL but with eventual loss of efficacy. A spectrum of subclonal genetic changes associated with VEN resistance has now been described. To fully understand clinical resistance to VEN, we combined single-cell short- and long-read RNA-sequencing to reveal the previously unappreciated scale of genetic and epigenetic changes underpinning acquired VEN resistance. These appear to be multilayered. One layer comprises changes in the BCL2 family of apoptosis regulators, especially the prosurvival family members. This includes previously described mutations in BCL2 and amplification of the MCL1 gene but is heterogeneous across and within individual patient leukemias. Changes in the proapoptotic genes are notably uncommon, except for single cases with subclonal losses of BAX or NOXA. Much more prominent was universal MCL1 gene upregulation. This was driven by an overlying layer of emergent NF-κB (nuclear factor kappa B) activation, which persisted in circulating cells during VEN therapy. We discovered that MCL1 could be a direct transcriptional target of NF-κB. Both the switch to alternative prosurvival factors and NF-κB activation largely dissipate following VEN discontinuation. Our studies reveal the extent of plasticity of CLL cells in their ability to evade VEN-induced apoptosis. Importantly, these findings pinpoint new approaches to circumvent VEN resistance and provide a specific biological justification for the strategy of VEN discontinuation once a maximal response is achieved rather than maintaining long-term selective pressure with the drug.
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ItemDescription of a novel subtype of acute myeloid leukemia defined by recurrent CBFB insertions(AMER SOC HEMATOLOGY, 2023-02-16)
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ItemNo Preview AvailableAcquired mutations in BAX confer resistance to BH3-mimetic therapy in acute myeloid leukemia(AMER SOC HEMATOLOGY, 2023-02-09)Randomized trials in acute myeloid leukemia (AML) have demonstrated improved survival by the BCL-2 inhibitor venetoclax combined with azacitidine in older patients, and clinical trials are actively exploring the role of venetoclax in combination with intensive chemotherapy in fitter patients with AML. As most patients still develop recurrent disease, improved understanding of relapse mechanisms is needed. We find that 17% of patients relapsing after venetoclax-based therapy for AML have acquired inactivating missense or frameshift/nonsense mutations in the apoptosis effector gene BAX. In contrast, such variants were rare after genotoxic chemotherapy. BAX variants arose within either leukemic or preleukemic compartments, with multiple mutations observed in some patients. In vitro, AML cells with mutated BAX were competitively selected during prolonged exposure to BCL-2 antagonists. In model systems, AML cells rendered deficient for BAX, but not its close relative BAK, displayed resistance to BCL-2 targeting, whereas sensitivity to conventional chemotherapy was variable. Acquired mutations in BAX during venetoclax-based therapy represent a novel mechanism of resistance to BH3-mimetics and a potential barrier to the long-term efficacy of drugs targeting BCL-2 in AML.
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ItemEnrichment of BTK Leu528Trp mutations in patients with CLL on zanubrutinib: potential for pirtobrutinib cross-resistance(ELSEVIER, 2022-10-25)The covalent Bruton's tyrosine kinase inhibitors (BTKis) are highly effective for the treatment of chronic lymphocytic leukemia (CLL). The dominant resistance mechanism observed with the BTKi ibrutinib is the development of BTK Cys481 codon mutations. Whether a similar resistance mutation profile exists for the newer-generation, more selective BTKi zanubrutinib is unknown. In samples referred for diagnostic next-generation sequencing in patients with progressive CLL, we observed an enrichment in the kinase-dead BTK Leu528Trp mutation in patients treated with zanubrutinib compared with ibrutinib (54%; 7 of 13 vs 4%; 1 of 24, P = .001). We describe 2 patients with BTK Leu528Trp mutations who showed clinical cross-resistance and progressive enrichment of the BTK Leu528Trp mutation over time when treated with the noncovalent BTKi pirtobrutinib. Both patients subsequently responded to venetoclax-based treatment. In summary, we have identified an enrichment of the BTK Leu528Trp mutation arising in patients treated with zanubrutinib that may impart cross-resistance to the noncovalent inhibitor pirtobrutinib and therefore may have implications for sequencing of these treatments in CLL.
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ItemSingle-cell sequencing demonstrates complex resistance landscape in CLL and MCL treated with BTK and BCL2 inhibitors(ELSEVIER, 2022-01-25)The genomic landscape of resistance to targeted agents (TAs) used as monotherapy in chronic lymphocytic leukemia (CLL) is complex and often heterogeneous at the patient level. To gain insight into the clonal architecture of acquired genomic resistance to Bruton tyrosine kinase (BTK) inhibitors and B-cell lymphoma 2 (BCL2) inhibitors in CLL, particularly in patients carrying multiple resistance mutations, we performed targeted single-cell DNA sequencing of 8 patients who developed progressive disease (PD) on TAs (either class). In all cases, analysis of single-cell architecture revealed mutual exclusivity between multiple resistance mutations to the same TA class, variable clonal co-occurrence of multiple mutations affecting different TAs in patients exposed to both classes, and a phenomenon of multiple independent emergences of identical nucleotide changes leading to canonical resistance mutations. We also report the first observation of established BCL2 resistance mutations in a patient with mantle cell lymphoma (MCL) following PD on sequential monotherapy, implicating BCL2 as a venetoclax resistance mechanism in MCL. Taken together, these data reveal the significant clonal complexity of CLL and MCL progression on TAs at the nucleotide level and confirm the presence of multiple, clonally independent, mechanisms of TA resistance within each individual disease context.
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ItemNo Preview AvailableClonal hematopoiesis, myeloid disorders and BAX-mutated myelopoiesis in patients receiving venetoclax for CLL(AMER SOC HEMATOLOGY, 2022-02-24)The BCL2 inhibitor venetoclax has established therapeutic roles in chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). As BCL2 is an important determinant of survival of both myeloid progenitor and B cells, we investigated whether clinical and molecular abnormalities arise in the myeloid compartment during long-term continuous venetoclax treatment of CLL in 89 patients (87 with relapsed/refractory CLL). Over a median follow-up of 75 (range 21-98) months, persistent cytopenias (≥1 of neutropenia, thrombocytopenia, anemia) lasting ≥4 months and unrelated to CLL occurred in 25 patients (28%). Of these patients, 20 (80%) displayed clonal hematopoiesis, including 10 with therapy-related myeloid neoplasms (t-MNs). t-MNs occurred exclusively in patients previously exposed to fludarabine-alkylator combination therapy with a cumulative 5-year incidence of 10.4% after venetoclax initiation, consistent with rates reported for patients exposed to fludarabine-alkylator combination therapy without venetoclax. To determine whether the altered myelopoiesis reflected the acquisition of mutations, we analyzed samples from patients with no or minimal bone marrow CLL burden (n = 41). Mutations in the apoptosis effector BAX were identified in 32% (13/41). In cellular assays, C-terminal BAX mutants abrogated outer mitochondrial membrane localization of BAX and engendered resistance to venetoclax killing. BAX-mutated clonal hematopoiesis occurred independently of prior fludarabine-alkylator combination therapy exposure and was not associated with t-MNs. Single-cell sequencing revealed clonal co-occurrence of mutations in BAX with DNMT3A or ASXL1. We also observed simultaneous BCL2 mutations within CLL cells and BAX mutations in the myeloid compartment of the same patients, indicating lineage-specific adaptation to venetoclax therapy.
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ItemNo Preview AvailableOutcomes of patients with CLL sequentially resistant to both BCL2 and BTK inhibition(ELSEVIER, 2021-10-26)Covalent Bruton tyrosine kinase inhibitors (BTKi's) and the B-cell lymphoma 2 (BCL2) inhibitor venetoclax have significantly improved outcomes for patients with chronic lymphocytic leukemia (CLL), especially those with biologically adverse disease. Patients with CLL resistant to their first targeted agent (TA) can be effectively treated with the alternative class. However, relapses are expected with second-line TA therapy, and the clinical challenge of double class-resistant disease is now emerging with increasing frequency. To define the characteristics and outcomes of patients with double class-resistant disease, we retrospectively analyzed 17 patients who developed progressive disease (PD) on both TA classes for CLL (venetoclax, then BTKi, n=12; BTKi, then venetoclax, n = 5). The cohort was heavily pretreated (median lines of prior therapy, 4) and enriched for adverse disease genetics (complex karyotype, 12 of 12 tested [100%]; del(17p)/TP53 mutations, 15 of 17 [88%]). The median time to progression on prior venetoclax was 24 months (range, 6-94 months) and was 25 months (range, 1-55 months) on prior BTKi. Progression on second-line TA was manifest as progressive CLL in 11 patients and as Richter transformation in 6. The median overall survival after progression on second-line TA was 3.6 months (95% confidence interval, 2-11 months). Patients with double class-resistant CLL have a dismal prognosis, representing a group of high unmet need.
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