Selective Inhibition Of Bcl-2 Research Articles

Cell selective BCL-2 inhibition enabled by lipid nanoparticles alleviates lung fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with a high mortality rate due to limited treatment options. Current therapies cannot effectively reverse the damage caused by IPF. Research suggests that promoting programmed cell death (apoptosis) in myofibroblasts, the key cells driving fibrosis, could be a promising strategy. However, inducing apoptosis in healthy cells like epithelial and endothelial cells can cause unwanted side effects. This project addresses this challenge by developing a targeted approach to induce apoptosis specifically in myofibroblasts. We designed liposomes (LPS) decorated with peptides that recognize VCAM-1, a protein highly expressed on myofibroblasts in fibrotic lungs. These VCAM1-targeted LPS encapsulate Venetoclax (VNT), a small molecule drug that inhibits BCL-2, an anti-apoptotic protein. By delivering VNT directly to myofibroblasts, we hypothesize that VCAM1-VNT-LPS can selectively induce apoptosis in these cells, leading to reduced fibrosis and improved lung function. We successfully characterized VCAM1-VNT-LPS for size, surface charge, and drug loading efficiency. Additionally, we evaluated their stability over three months at different temperatures. In vitro and in vivo studies using a bleomycin-induced mouse model of lung fibrosis demonstrated the therapeutic potential of VCAM1-VNT-LPS. These studies showed a reduction in fibrosis-associated proteins (collagen, α-SMA, VCAM1) and BCL-2, while simultaneously increasing apoptosis in myofibroblasts. These findings suggest that VCAM1-targeted delivery of BCL-2 inhibitors using liposomes presents a promising and potentially selective therapeutic approach for IPF.

Dihydroartemisinin Synergistically Promotes the Antileukemic Activity of Venetoclax By Downregulating Mcl-1 in Acute Myeloid Leukemia Cells

Background: Venetoclax is a BH3 mimetic, which selectively inhibits Bcl-2. For older persons with newly diagnosed AML, the FDA has approved the use of venetoclax in conjunction with hypomethylating drugs or low-dose cytarabine. However, the selective inhibition of Bcl-2 in AML often results in the overexpression of Mcl-1, another dominant anti-apoptotic Bcl-2 family protein conferring venetoclax resistance. Dihydroartemisinin (DHA) is a herbal monomer that is taken orally to cure malaria. Studies have shown that DHA downregulates MCL-1 protein expression in AML cells. In the present study, we investigated whether the combination of venetoclax and DHA efficiently promotes apoptosis in AML cells. Methods: We assessed the combined effect of venetoclax and DHA on cell viability, apoptosis, combination index, and cell cycle in AML cells from AML cell lines and deno AML patients. Western blot analysis was used to validate the mechanisms of venetoclax and DHA. Results: Venetoclax efficiently impaired cell viability and dose-dependently promoted apoptosis when combined with DHA; their synergism was aptly represented by the combination index. Venetoclax and DHA had significant synergistic inhibition of cell viability both in cells from AML cell lines and in deno AML patients. The combination of venetoclax and DHA impaired cell cycle progression by arresting the cell cycle in S-phase and inducing apoptosis. Mechanistically, DHA mitigated venetoclax-induced upregulation of Mcl-1 and BCL-XL. Moreover, the combination inhibited the expression of C-MYC. Conclusions: Our findings demonstrate the synergistic, preferential antileukemic effects of venetoclax and DHA on AML cells, providing a rationale for preclinical and clinical trials by combining these agents already being used in clinical practice to treat AML.

Research Highlights.

Research Highlights.

Selective Bcl-2 inhibition promotes hematopoietic chimerism and allograft tolerance without myelosuppression in nonhuman primates.

Hematopoietic stem cell transplantation (HSCT) has many potential applications beyond current standard indications, including treatment of autoimmune disease, gene therapy, and transplant tolerance induction. However, severe myelosuppression and other toxicities after myeloablative conditioning regimens have hampered wider clinical use. To achieve donor hematopoietic stem cell (HSC) engraftment, it appears essential to establish niches for the donor HSCs by depleting the host HSCs. To date, this has been achievable only by nonselective treatments such as irradiation or chemotherapeutic drugs. An approach that is capable of more selectively depleting host HSCs is needed to widen the clinical application of HSCT. Here, we show in a clinically relevant nonhuman primate model that selective inhibition of B cell lymphoma 2 (Bcl-2) promoted hematopoietic chimerism and renal allograft tolerance after partial deletion of HSCs and effective peripheral lymphocyte deletion while preserving myeloid cells and regulatory T cells. Although Bcl-2 inhibition alone was insufficient to induce hematopoietic chimerism, the addition of a Bcl-2 inhibitor resulted in promotion of hematopoietic chimerism and renal allograft tolerance despite using only half of the dose of total body irradiation previously required. Selective inhibition of Bcl-2 is therefore a promising approach to induce hematopoietic chimerism without myelosuppression and has the potential to render HSCT more feasible for a variety of clinical indications.

Abstract 1631: Combination of olverembatinib (HQP1351) with BCL-2 inhibitor lisaftoclax (APG-2575) overcomes resistance in gastrointestinal stromal tumors (GISTs)

Abstract GISTs are common malignant mesenchymal tumors that occur in the GI tract, with an estimated incidence rate of 1% to 2%. About 75% to 80% of patients with GIST have mutations in the KIT gene, while 5% to 10% have mutations in the platelet-derived growth factor receptor α (PDGFRA) gene. Tyrosine kinase inhibitors (TKIs) offer patients an improved quality of life but increased pharmacological resistance to TKIs is often observed. BCL-2 is expressed in >80% of GISTs, and amplification of BCL-2 and BCL-xL are common features associated with disease progression. One approach to enhance GIST eradication is to concurrently inhibit oncogenic KIT signaling while actively engaging apoptotic pathways. Olverembatinib (HQP1351) is a new, third-generation TKI that targets BCR-ABL1, KIT and PDGFRA and is currently in development for relapsed or refractory chronic myeloid leukemia and GIST. Lisaftoclax (APG-2575) is a selective BCL-2 inhibitor under development for hematologic malignancies. The purpose of this study was to evaluate whether combining a BCL-2 inhibitor, lisaftoclax, with olverembatinib enhances treatment effect on imatinib-resistant GISTs. Compared to imatinib-sensitive GIST cell lines (GIST882 and GIST-T1), increased BCL-2 protein expression was observed in imatinib-resistant GIST cells, particularly in GIST430, GIST48, and GIST48B lines. Cell-based antiproliferation studies showed additive activity of olverembatinib and lisaftoclax in imatinib-resistant GIST cells. In vivo studies using imatinib-resistant GIST430 cell-derived xenograft models further revealed that coadministration of olverembatinib at 15 mg/kg (every other day for 3 weeks) with lisaftoclax at 50 mg/kg (daily for 3 weeks) resulted in enhanced antitumor effects compared to either agent alone. Tumor growth inhibition (TGI) rates for combined treatment with olverembatinib and lisaftoclax reached 76.8%, which was superior to the TGI rate of either agent alone: 57.6% and 31.2% for olverembatinib and lisaftoclax, respectively. Regarding putative mechanisms, olverembatinib treatment was shown to downregulate BCL-xL and MCL-1 protein expression, possibly via inhibition of KIT signal pathway and downstream signal transducer and activator of transcription 3 (STAT3). Lisaftoclax triggered apoptosis by disrupting the BCL-2:BIM complex. Therefore, selective inhibition of BCL-2, aided by TKI-mediated MCL-1 and BCL-XL inhibition, markedly augmented cleavage of caspase-3 and poly (ADP-ribose) polymerase 1 (PARP-1), thus triggering apoptosis and subsequently enhancing antitumor effects. Our results demonstrate that olverembatinib and BCL-2 inhibitor lisaftoclax have additive antitumor effects in imatinib-resistant GIST. This novel dual approach may have the potential for treating patients with GISTs whose disease has progressed after treatment with TKIs. Citation Format: Ping Min, Guangfeng Wang, Eric Liang, Bingxing Wu, Huidan Yu, Dajun Yang, Yifan Zhai. Combination of olverembatinib (HQP1351) with BCL-2 inhibitor lisaftoclax (APG-2575) overcomes resistance in gastrointestinal stromal tumors (GISTs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1631.

Management of Relapsed/Refractory Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma in the Era of Targeted Therapies.

The treatment landscape for relapsed chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) has changed substantially over the past decade and continues to evolve. Despite the emergence of targeted therapies that are well tolerated and prolong survival, the disease remains incurable and relapse is common particularly in individuals with high-risk features. Herein, we review the key literature about the current options for relapsed disease and explore the emerging role of cellular therapies. Clinical trials have established the role of Bruton tyrosine kinase inhibitors, selective BCL-2 inhibition, and anti-CD20 monoclonal antibodies as treatment options for CLL/SLL. The role of chimeric antigen receptor T cells has shown promise in individuals with CLL/SLL in early phase clinical trials. Novel therapeutic approaches with targeted therapies have redefined the management of CLL/SLL in both the front-line and relapsed/refractory settings. Optimal management in terms of sequencing or combining therapies, especially in individuals with high-risk features, remains a challenge. The emerging role of cellular therapies has the potential to build upon and further improve the current treatment paradigm.

Arsenic trioxide synergistically promotes the antileukaemic activity of venetoclax by downregulating Mcl-1 in acute myeloid leukaemia cells

BackgroundThe evasion of apoptosis through dysregulated Bcl-2 family members is a hallmark of leukaemia stem cells (LSCs) in acute myeloid leukaemia (AML). Therefore, targeting Bcl-2 with venetoclax has been suggested as an attractive strategy for inducing apoptosis in AML LSCs. However, the selective inhibition of Bcl-2 in AML often leads to upregulation of Mcl-1, another dominant anti-apoptotic Bcl-2 family protein conferring venetoclax resistance.MethodsWe assessed the combined effect of venetoclax and arsenic trioxide (ATO) on leukaemic cell viability, apoptosis, combination index, and cell cycle in the human LSC-like KG1 and KG1a cells. The synergistic effect of venetoclax and ATO on apoptosis was also examined in primary CD34+ and CD34+CD38− LSCs from the bone marrow (BM) of AML patients, and compared with those from healthy donors.ResultsVenetoclax efficiently impaired cell viability and dose-dependently promoted apoptosis when combined with ATO; their synergism was aptly represented by the combination index. The combination of venetoclax and ATO impaired cell cycle progression by restricting cells within the sub-G1 phase and facilitating caspase-dependent apoptotic cell death associated with the loss of mitochondrial membrane potential, while sparing healthy BM haematopoietic stem cells. Mechanistically, ATO mitigated venetoclax-induced upregulation of Mcl-1 by the inhibition of AKT and ERK, along with activation of GSK-3β. This led to the Mcl-1 destabilisation, triggering Noxa and Bim to facilitate apoptosis and the consequent activation of the apoptosis executioner protein Bak. Moreover, the combination promoted phosphorylation of ATM, Chk2, p38, and H2AX, indicating an active DNA damage response.ConclusionsOur findings demonstrate the synergistic, preferential antileukaemic effects of venetoclax and ATO on LSCs, providing a rationale for preclinical and clinical trials by combining these agents already being used in clinical practice to treat acute leukaemia.

Dasatinib Stimulates Its Own Mechanism of Resistance by Activating a CRTC3/MITF/Bcl-2 Pathway in Melanoma with Mutant or Amplified c-Kit.

Amplification or activating mutations of c-Kit are a frequent oncogenic alteration, which occurs commonly in acral and mucosal melanoma. Among c-Kit inhibitors, dasatinib is the most active due to its ability to bind both active and inactive conformations of the receptor. However, its use as a single agent in melanoma showed limited clinical benefit. We first found that sensitivity to dasatinib is restricted to melanoma cell lines harboring c-Kit alteration but, unexpectedly, we observed lower effect at higher concentrations that can readily be found in patient blood. We then investigated relevant pathway alterations and found complete inhibition of MAPK and PI3K/AKT pathways but an increase in MITF and its downstream target Bcl-2 through CRTC3 pathway, which turn on the CREB regulated transcription of MITF. More importantly, dasatinib upregulates MITF and Bcl-2 through SIK2 inhibition revealed by CRTC3 reduced phosphorylation, CREB transcription activation of MITF, MITF transcription activation of Bcl-2 as well as pigmentation. Furthermore, overexpression of MITF renders melanoma cells resistant to all dasatinib concentrations. Selective Bcl-2 inhibition by ABT-199 or Bcl-2 knockout restores the sensitivity of melanoma cells to dasatinib, validating the involvement of MITF and Bcl-2 axis in the resistance of melanoma to dasatinib. In conclusion, we showed for the first time that dasatinib in melanoma stimulates its proper mechanism of resistance, independently of MAPK and PI3K/AKT pathways reactivation commonly associated to secondary c-Kit mutations, but through CRTC3/MITF/Bcl-2 pathway activation at clinically relevant doses which may explain the weak clinical benefit of dasatinib in patients with melanoma. IMPLICATIONS: Dasatinib stimulates its proper mechanism of resistance through CRTC3/MITF/Bcl-2 pathway, which may explain its modest clinical efficiency in patients with melanoma.

Dual targeting of BCL2 and MCL1 rescues myeloma cells resistant to BCL2 and MCL1 inhibitors associated with the formation of BAX/BAK hetero-complexes

Multiple myeloma is a plasma cell malignancy that escapes from apoptosis by heterogeneously over-expressing anti-apoptotic BCL2 proteins. Myeloma cells with a t(11;14) translocation present a particular vulnerability to BCL2 inhibition while a majority of myeloma cells relies on MCL1 for survival. The present study aimed to determine whether the combination of BCL2 and MCL1 inhibitors at low doses could be of benefit for myeloma cells beyond the single selective inhibition of BCL2 or MCL1. We identified that half of patients were not efficiently targeted neither by BCL2 inhibitor nor MCL1 inhibitor. Seventy percent of these myeloma samples, either from patients at diagnosis or relapse, presented a marked increase of apoptosis upon low dose combination of both inhibitors. Interestingly, primary cells from a patient in progression under venetoclax treatment were not sensitive ex vivo to neither venetoclax nor to MCL1 inhibitor, whereas the combination of both efficiently induced cell death. This finding suggests that the combination could overcome venetoclax resistance. The efficacy of the combination was also confirmed in U266 xenograft model resistant to BCL2 and MCL1 inhibitors. Mechanistically, we demonstrated that the combination of both inhibitors favors apoptosis in a BAX/BAK dependent manner. We showed that activated BAX was readily increased upon the inhibitor combination leading to the formation of BAK/BAX hetero-complexes. We found that BCLXL remains a major resistant factor of cell death induced by this combination. The present study supports a rational for the clinical use of venetoclax/S63845 combination in myeloma patients with the potential to elicit significant clinical activity when both single inhibitors would not be effective but also to overcome developed in vivo venetoclax resistance.

Mutations and Copy Number Gains of the BCL2 Family Members Mediate Resistance to Venetoclax in Multiple Myeloma (MM) Patients

Background: Selective BCL2 inhibition with venetoclax, induces deep responses in relapsed MM. However, acquired resistance to venetoclax frequently occurs. Importantly, in the Bellini trial, while the combination of venetoclax and bortezomib doubled the median progression free survival compared to control, it resulted in shorter overall survival. These results highlight the challenges secondary resistance poses and the need to investigate the mechanisms mediating the emerged resistance to venetoclax. Methods & Results: Bone marrow (BM) aspirates were collected from patients (n=8) treated with venetoclax prior to initiation of therapy and at disease progression. Mononuclear cell fractions were isolated through ficoll coupled with magnetic sorting of CD138+cells. Ex-vivo functional profiling of venetoclax sensitivity was performed on CD138+cells. Unbiased mRNA and DNA profiling was conducted by single-cell RNA-sequencing (scRNA-seq), single cell ATAC-seq and single cell copy number analysis (scCNV) using the GemCode system (10x Genomics). Cell Ranger, Seurat, Signac and Monocle were used for data analysis. Ex-vivo apoptosis studies revealed a dynamic shift of the IC50s of venetoclax from a median of 100 nM in pretreatment sample to >1000 nM at disease progression. Single cell CNV profiling identified a significant expansion of a pre-treatment subclonal (<1%) cluster of cells with 1q21 copy number gain to become the predominant clone (>70%) at the time of relapse. The scale of the 1q CNV gain varied from 3 Mb to a very focal gain of 100 kb encompassing the MCL1 gene locus. Of note copy number gain was also identified in the BCL2L1 gene, but no copy number losses were detected in the BAX or BAK gene loci. Single cell RNA profiling of the same samples confirmed the gain in the MCL1 mRNA transcript with downregulation of BCL2 at the time of relapse. Consistent with a branching evolutionary model, scATAC-seq revealed increased chromatin accessability studies at the MCL1 locus in subclones of relapsed samples. Cell trajectory analysis and pseudotime ordering of cells (Monocle) revealed the emergence of a highly proliferative clone and of an MCL1 dependent clone as the disease evolved from its original BCL2 dependent cluster at pseudotime (t0) (Figure). Furthermore, ex-vivo apoptosis profiling revealed a shift in the sensitivity of the CD138+cells with an acquired sensitivity to MCL1 inhibitor (S63845). These findings suggest that the 1q21 copy number gain with MCL1 upregulation is likely mediating the acquired venetoclax resistance. In order to functionally confirm whether the gain in MCL1 is sufficient to shift BCL2 to MCL1 dependency and induce resistant to venetoclax, we stably overexpressed MCL1 in the KMS12PE BCL2-dependent MM cell line (KMS12PE_MCL1) and examined its sensitivity to venetoclax and/or S63845 relative to control vector (KMS12PE_EV). Of interest, co-immunoprecipitation (co-IP) of BIM in KMS12PE_MCL1 demonstrated a shift in BIM loading and co-IP with MCL1 and BCL2 while it was restricted to BCL2 in KMS12PE_EV cells. Importantly, venetoclax IC50 of KMS12PE_EV increased by 200 folds in KMS12PE_MCL1 cells with acquired sensitivity to S63845. scCNV analysis of one patient did not identify any gain in the 1q locus at the time of disease progression to venetoclax. Mutation analysis however identified a de novo BCL2 mutation [NM_000633.2:c.332A>C, p.(Asp111Ala)]. To determine whether the Asp111Ala mutation alone is sufficient to confer resistance to venetoclax, the mutant was overexpressed in KMS12PE cells. While BIM binding to BCL2 was unaffected, Aps111Ala largely abrogated venetoclax-induced BIM displacement from BCL2 and reduced KMS12PE cells sensitivity to venetoclax by ~7.5 folds. Structure modeling also demonstrated the inability of venotoclax to tightly bind mutant BCL2_Asp111Ala hydrophobic groove. Conclusion: We have discovered and functionally characterized a novel BCL2 mutation that confers in vitro and in vivo resistance to venetoclax-treated MM patients. In addition we have identified through single cell profiling an enrichment of MM clones with MCL1 locus copy number gain at the time of acquired venetoclax resistance. Early detection and dynamic monitoring of these abnormalities (BCL2 mutant or 1q gain) with early therapeutic interventions targeting these clones may enhance venetoclax efficacy and improve patients' survival. Figure Disclosures Neri: Celgene, Janssen: Consultancy, Honoraria, Research Funding. Boise:Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Bahlis:Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria.

Nutritional preconditioning induced by astragaloside Ⅳ on isolated hearts and cardiomyocytes against myocardial ischemia injury via improving Bcl-2-mediated mitochondrial function

Ischemic preconditioning and pharmacological preconditioning are common strategies to prevent lethal myocardial injury, especially nutritional preconditioning (NPC). In this study, we investigated the effects of astragaloside IV (Ast), as an NPC agent, on myocardium suffered anoxia/reoxygenation (A/R) injury. Rats received 5 mg/kg Ast daily for 3 weeks by intragastric administration. Then, hearts were harvested and underwent A/R treatment using a Langendorff apparatus. Ast- pretreatment significantly promoted functional recovery of the myocardium, reduced infarct size, and oxidative stress, and decreased the apoptotic index. Similar findings were demonstrated in H9c2 cardiomyocytes that were pretreated with Ast for 24 h. Moreover, Ast-pretreatment significantly upregulated Bcl-2 expression, especially in mitochondria. The effects of Ast treatment against A/R injury were also reflected by increased antioxidant potential, inhibited reactive oxygen species (ROS) burst, increased oxygen consumption rate, maintained mitochondrial membrane potential (MMP), inhibited mitochondrial permeability transition pore (mPTP) opening, and prevented apoptosis. Selective inhibition of Bcl-2 by ABT-737 decreased myocardial injury protection of Ast. Ast-pretreatment resulted in NPC- related effects against A/R, and mitochondria may be the target of a cascade of events elicited by upregulating Bcl-2 expression, promoting translocation of Bcl-2 into mitochondria, maintaining MMP, inhibiting ROS bursts, thereby leading to recovery of mitochondrial respiration, preventing mPTP opening, decreasing cytochrome C release, preventing apoptosis, and ultimately alleviating myocardial injury.

Interactions between cancer-associated fibroblasts and tumor cells promote MCL-1 dependency in estrogen receptor-positive breast cancers

Selective inhibition of BCL-2 is expected to enhance therapeutic vulnerability in luminal estrogen receptor-positive breast cancers. We show here that the BCL-2 dependency of luminal tumor cells is nevertheless mitigated by breast cancer-associated fibroblasts (bCAFs) in a manner that defines MCL-1 as another critical therapeutic target. bCAFs favor MCL-1 expression and apoptotic resistance in luminal cancer cells in a IL-6 dependent manner while their own, robust, survival also relies on MCL-1. Studies based on ex vivo cultures of human luminal breast cancer tissues further argue that the contribution of stroma-derived signals to MCL-1 expression shapes BCL-2 dependency. Thus, MCL-1 inhibitors are beneficial for targeted apoptosis of breast tumor ecosystems, even in a subtype where MCL-1 dependency is not intrinsically driven by oncogenic pathways.

Cell-Type Specific Mechanisms of Hematopoietic Stem Cell (HSC) Expansion Underpin Progressive Disease in Myelodysplastic Syndromes (MDS) and Provide a Rationale for Targeted Therapies

The mechanisms of HMA failure in MDS remain unclear, as recent advances in sequencing approaches did not enable the molecular characterization of the cells that survive therapy and drive resistance and disease progression.Here, through combined functional and transcriptomic analyses of highly-purified hematopoietic populations isolated from the BM of 132 MDS patients enrolled in clinical trials of single drug HMA therapy, we show that 2 immunophenotypically and molecularly distinct cell types maintain the disease and expand at progression, and we propose therapeutic approaches to overcome MDS evolution.Unsupervised hierarchical clustering followed by principal component analysis of 101 untreated MDS samples based on the frequency of immunophenotypically defined stem and myeloid progenitor cell populations identified the frequencies of the lymphoid-primed multipotent progenitors (LMPPs) and granulo-monocytic progenitors (GMPs) as the main sources of variation across the samples. Further logistic regression analysis enabled the systematic stratification of the samples in 2 main groups. “CMP pattern” MDS was characterized by the prevalence of common myeloid progenitors (CMPs) in the progenitor compartment while “GMP pattern” MDS was characterized by the increased frequency of GMPs in the progenitors and by increased LMPPs and decreased long-term (LT)-HSC frequencies in the BM (Fig A, B). Functional analysis of the CMPs by immunophenotypic characterization of lineage-primed fractions and colony assays revealed that this population was myeloid-biased only in “CMP pattern” MDS but not in “GMP pattern” MDS, suggesting that 2 distinct hierarchical differentiation routes underlie disease manifestation (Fig C). Further logistic regression analysis of 31 MDS samples from patients with progressive disease showed that whereas “CMP pattern” MDS patients with blast expansion were characterized by a significant increase in the BM frequency of LT-HSCs, “GMP pattern” MDS patients were characterized by the expansion of the LMPPs (Fig D).To investigate the molecular pathways underlying the 2 different hierarchical patterns of progression, we analyzed the transcriptional profiling of the HSCs that selectively expanded in the 2 groups of MDS patients. RNA-Seq analysis revealed that, compared with those isolated from patients at baseline, LT-HSCs isolated from “CMP pattern” MDS patients with blast expansion had significantly upregulated genes involved in promoting cell proliferation and survival, including the anti-apoptotic regulator BCL2. In contrast, genes involved in the response to TNF-a were significantly upregulated in the LMPPs from “GMP pattern” MDS patients with progressive disease as compared with the LMPPs isolated at baseline (Fig E). Then, we hypothesized that, despite genetic dissimilarities, the HSCs that expanded at progression were addicted to the molecular pathways that were upregulated and that targeting these pathways represented a potential therapeutic strategy to overcome HMA resistance. As proof-of-principle, we treated CD34+ cells from “CMP pattern” MDS isolated after HMA failure, co-cultured over a layer of stromal cells, with the BCL2 inhibitor ABT-199 for 72 hours. ABT-199, in combination with 5-azacytidine (AZA), significantly decreased the number of LT-HSCs from MDS patients with progressive disease (Fig F), but did not affect those from MDS patients in whom HMA therapy failed because of persistent MDS. These data suggest that ABT-199 selectively targets the LT-HSCs from “CMP pattern” MDS with progressive disease, which depend on increased levels of BCL2 to support their expansion. Treatment of WT/vav-cre-TET2L/L BM chimeras with ABT-199 therapy showed selective apoptosis and depletion of the MDS-like LT-HSCs and the concomitant proliferation of the WT LT-HSCs. This suggests that ABT-199 allows selective therapeutic targeting that eliminates abnormal HSCs while restoring normal hematopoiesis (Fig G). Furthermore, ABT-199 treatment of xenografts generated by transplanting the MDS-L cell line inNSGS mice reduced tumoral burden by depleting the human blast population (Fig H).Taken together, these findings demonstrate that targeting commonly deregulated pathways in MDS HSCs is a feasible approach to tackling disease progression and provide a rationale for the selective inhibition of BCL2 in “CMP pattern” MDS with progressive disease. [Display omitted] DisclosuresGiuliani:Celgene Italy: Other: Avisory Board, Research Funding; Takeda Pharmaceutical Co: Research Funding; Janssen Pharmaceutica: Other: Avisory Board, Research Funding. Konopleva:Stemline Therapeutics: Research Funding. Colla:Abbvie: Research Funding.

Bcl-xl as the most promising Bcl-2 family member in targeted treatment of chondrosarcoma

Chondrosarcomas are malignant cartilage tumors showing relative resistance to conventional chemo- and radiotherapy. Previous studies showed that chondrosarcoma cells could be sensitized to chemotherapy by inhibiting the Bcl-2 family members Bcl-2, Bcl-xl and Bcl-w using ABT-737. In this study we explored the specific role of Bcl-2 family members to identify the most important player in chondrosarcoma cell survival and chemo resistance. Immunohistochemistry was performed on tissue microarrays containing 137 conventional chondrosarcomas of different grades. Selective inhibition of Bcl-2 (S55746) or Bcl-xl (WEHI-539 or A-1155463) and the combination with doxorubicin or cisplatin was investigated in a panel of 8 chondrosarcoma cell lines using presto blue viability assays and caspase 3/7 glo apoptosis assays. In addition Bcl-2 and Bcl-xl inhibition was investigated in an orthotopic Swarm Rat Chondrosarcoma (SRC) model. Bcl-2 and Bcl-xl were most abundantly expressed in the primary tumors, and expression increased with increasing histological grade. A subset of chondrosarcoma cell lines was sensitive to selective inhibition of Bcl-xl, and synergy was observed with doxorubicin or cisplatin in 3 out of 8 chondrosarcoma cell lines resulting in apoptosis. Conversely, selective inhibition of Bcl-2 was not effective in chondrosarcoma cell lines and could not sensitize to chemotherapy. In vivo, selective inhibition of Bcl-xl, but not Bcl-2 resulted in a decrease in tumor growth rate, even though no sensitization to doxorubicin was observed. These results suggest that among the Bcl-2 family members, Bcl-xl is most important for chondrosarcoma survival. Further research is needed to validate whether single or combination treatment with chemotherapy will be beneficial for chondrosarcoma patients.

Identification of molecular features necessary for selective inhibition of B cell lymphoma proteins using machine learning techniques

Selective inhibition of Bcl-2 and Bcl-xL proteins due to their dual inhibition toxicity plays an important role in treatment of cancer and chemotherapy effectiveness; therefore, in the last decade, discovery of selective inhibitors for Bcl-2 and Bcl-xL proteins has become a significant and important research topic. The present contribution paves the way for characterization of molecular features which induce selectivity for inhibition of Bcl-2 and Bcl-xL. In this line, a total of 1534 molecules related to inhibition of Bcl-2 and Bcl-xL proteins were collected from Binding Database. A diverse set of molecular descriptors was calculated for each molecule, and the best subset of descriptors were selected using variable importance in projection (VIP) approach. The molecules were classified according to their therapeutic targets (Bcl-2/Bcl-xL) and activities. Partial least square-discriminate analysis (PLS-DA) and supervised Kohonen network (SKN) models were utilized to relate the molecular structures of chemicals to their activities and selectivities. According to the VIP-selected descriptors physicochemical properties, such as polarity number, number of branches, size and cyclicity of the molecule, flexibility, functional counts and constitutional descriptors, all affect the activities of Bcl-2 and Bcl-xL inhibitors. The performances of PLS-DA and SKN methods were evaluated based on statistical parameters derived from the confusion matrices. The models were validated using tenfold cross-validation and an external test set. The best statistical results were obtained by implementing the SKN model. The classification rates range from 93.5 to 79.1% for the training and validation procedure for the optimized SKN models. The high values of the obtained classification rates demonstrate that the information provided in this work would be useful to design new drugs with selective inhibitory activities toward Bcl-2 or Bcl-xL proteins for more effective treatment of cancer.

Selective inhibition of BCL-2 is a promising target in patients with high-risk myelodysplastic syndromes and adverse mutational profile.

Somatic mutations in genes such as ASXL1, RUNX1, TP53 or EZH2 adversely affect the outcome of patients with myelodysplastic syndromes (MDS). Since selective BCL-2 inhibition is a promising treatment strategy in hematologic malignancies, we tested the therapeutic impact of ABT-199 on MDS patient samples bearing an adverse mutational profile. By gene expression, we found that the level of pro-apoptotic BIM significantly decreased during MDS disease progression in line with an acquired resistance to cell death. Supporting the potential for ABT-199 treatment in MDS, high-risk MDS patient samples specifically underwent cell death in response to ABT-199 even when harbouring mutations in ASXL1, RUNX1, TP53 or EZH2. ABT-199 effectively targeted the stem- and progenitor compartment in advanced MDS harbouring mutations in ASXL1, RUNX1, TP53 or EZH2 and even proved effective in patients harbouring more than one of the defined high-risk mutations. Moreover, we utilized the protein abundance of BCL-2 family members in primary patient samples using flow cytometry as a biomarker to predict ABT-199 treatment response. Our data demonstrate that ABT-199 effectively induces apoptosis in progenitors of high-risk MDS/sAML despite the presence of adverse genetic mutations supporting the notion that pro-apoptotic intervention will hold broad therapeutic potential in high-risk MDS patients with poor prognosis.

P53 and MITF/Bcl-2 identified as key pathways in the acquired resistance of NRAS-mutant melanoma to MEK inhibition

Activating mutations in Neuroblastoma RAS viral oncogene homolog (NRAS) are found in 15–30% of melanomas and are associated with a poor prognosis. Although MAP kinase kinase (MEK) inhibitors used as single agents showed a limited clinical benefit in patients with NRAS-mutant melanoma due to their rather cytostatic effect and high toxicity, their combination with other inhibitors of pathways known to cooperate with MEK inhibition may maximise their antitumour activity. Similarly, in a context where p53 is largely inactivated in melanoma, hyperexpression of Microphthalmia associated transcription factor (MITF) and its downstream anti-apoptotic targets may be the cause of the restraint cytotoxic effects of MEK inhibitors. Indeed, drug combinations targeting both mutant BRAF and MITF or one of its important targets Bcl-2 were effective in mutant BRAF melanoma but had no effect on acquired resistance. Therefore, we aimed to further investigate the downstream MITF targets that can explain this anti-apoptotic effect and to evaluate in parallel the effect of p53 reactivation on the promotion of apoptosis under MEK inhibition in a panel of Q61NRAS-mutant melanoma cells. First, we showed that MEK inhibition (pimasertib) led to a significant inhibition of cell proliferation but with a limited effect on apoptosis that could be explained by the systematic MITF upregulation. Mimicking the MITF effect via cyclic adenosine monophosphate activation conferred resistance to MEK inhibition and upregulated Bcl-2 expression. In addition, acquired resistance to MEK inhibition was associated with a strong activation of the anti-apoptotic signalling MITF/Bcl-2. More importantly, selective Bcl-2 inhibition by ABT-199 or Bcl-2 knockout using CRISPR/Cas9 system annihilated the acquired resistance and restored the sensitivity of NRAS-mutant melanoma cells to MEK inhibition. Strikingly and similarly, direct p53 reactivation (PRIMA-1Met, APR-246) also broke resistance and synergised with MEK inhibition to induce massive apoptosis in NRAS-mutant melanoma cells with wild-type or mutant p53. Hence, our data identify MITF/Bcl-2 as a key mechanism underlying resistance of NRAS-mutant melanoma cells to apoptosis by MEK inhibitors and paves the way for a promising drug combination that could prevent or reverse anti-MEK resistance in this group of patients.

Maintenance of the HIV Reservoir Is Antagonized by Selective BCL2 Inhibition.

Decay of the HIV reservoir is slowed over time in part by expansion of the pool of HIV-infected cells. This expansion reflects homeostatic proliferation of infected cells by interleukin-7 (IL-7) or antigenic stimulation, as well as new rounds of infection of susceptible target cells. As novel therapies are being developed to accelerate the decay of the latent HIV reservoir, it will be important to identify interventions that prevent expansion and/or repopulation of the latent HIV reservoir. Our previous studies showed that HIV protease cleaves the host protein procaspase 8 to generate Casp8p41, which can bind and activate Bak to induce apoptosis of infected cells. In circumstances where expression of the anti-apoptotic protein BCL2 is high, Casp8p41 instead binds BCL2, and cell death does not occur. This effect can be overcome by treating cells with the clinically approved BCL2 antagonist venetoclax, which prevents Casp8p41 from binding BCL2, thereby allowing Casp8p41 to bind Bak and kill the infected cell. Here we assess whether the events that maintain the HIV reservoir are also antagonized by venetoclax. Using the J-Lat 10.6 model of persistent infection, we demonstrate that proliferation and HIV expression are countered by the use of venetoclax, which causes preferential killing of the HIV-expressing cells. Similarly, during new rounds of infection of primary CD4 T cells, venetoclax causes selective killing of HIV-infected cells, resulting in decreased numbers of HIV DNA-containing cells.IMPORTANCE Cure of HIV infection requires an intervention that reduces the HIV reservoir size. A variety of approaches are being tested for their ability to impact HIV reservoir size. Even if successful, however, these approaches will need to be combined with additional complementary approaches that prevent replenishment or repopulation of the HIV reservoir. Our previous studies have shown that the FDA-approved BCL2 antagonist venetoclax has a beneficial effect on the HIV reservoir size following HIV reactivation. Here we demonstrate that venetoclax also has a beneficial effect on HIV reservoir size in a model of homeostatic proliferation of HIV as well as in acute spreading infection of HIV in primary CD4 T cells. These results suggest that venetoclax, either alone or in combination with other approaches to reducing HIV reservoir size, is a compound worthy of further study for its effects on HIV reservoir size.

Venetoclax plus rituximab in relapsed or refractory chronic lymphocytic leukaemia: a phase 1b study

Selective BCL2 inhibition with venetoclax has substantial activity in patients with relapsed or refractory chronic lymphocytic leukaemia. Combination therapy with rituximab enhanced activity in preclinical models. The aim of this study was to assess the safety, pharmacokinetics, and activity of venetoclax in combination with rituximab. Adult patients with relapsed or refractory chronic lymphocytic leukaemia (according to the 2008 Modified International Workshop on CLL guidelines) or small lymphocytic lymphoma were eligible for this phase 1b, dose-escalation trial. The primary outcomes were to assess the safety profile, to determine the maximum tolerated dose, and to establish the recommended phase 2 dose of venetoclax when given in combination with rituximab. Secondary outcomes were to assess the pharmacokinetic profile and analyse efficacy, including overall response, duration of response, and time to tumour progression. Minimal residual disease was a protocol-specified exploratory objective. Central review of the endpoints was not done. Venetoclax was dosed daily using a stepwise escalation to target doses (200-600 mg) and then monthly rituximab commenced (375 mg/m2 in month 1 and 500 mg/m2 in months 2-6). Adverse events were graded according to the National Cancer Institute Common Terminology Criteria for adverse events version 4.0. Protocol-guided drug cessation was allowed for patients who achieved complete response (including complete response with incomplete marrow recovery) or negative bone marrow minimal residual disease. Analyses were done per protocol for all patients who commenced drug and included all patients who received at least one dose of venetoclax. Data were pooled across dose cohorts. Patients are still receiving therapy and follow-up is ongoing. The trial is registered at ClinicalTrials.gov, number NCT01682616. Between Aug 6, 2012, and May 28, 2014, we enrolled 49 patients. Common grade 1-2 toxicities included upper respiratory tract infections (in 28 [57%] of 49 patients), diarrhoea (27 [55%]), and nausea (25 [51%]). Grade 3-4 adverse events occurred in 37 (76%) of 49 patients; most common were neutropenia (26 [53%]), thrombocytopenia (eight [16%]), anaemia (seven [14%]), febrile neutropenia (six [12%]), and leucopenia (six [12%]). The most common serious adverse events were pyrexia (six [12%]), febrile neutropenia (five [10%]), lower respiratory tract infection, and pneumonia (each three [6%]). Clinical tumour lysis syndrome occurred in two patients (resulting in one death) who initiated venetoclax at 50 mg. After enhancing tumour lysis syndrome prophylaxis measures and commencing venetoclax at 20 mg, clinical tumour lysis syndrome did not occur. The maximum tolerated dose was not identified; the recommended phase 2 dose of venetoclax in combination with rituximab was 400 mg. Overall, 42 (86%) of 49 patients achieved a response, including a complete response in 25 (51%) of 49 patients. 2 year estimates for progression-free survival and ongoing response were 82% (95% CI 66-91) and 89% (95% CI 72-96), respectively. Negative marrow minimal residual disease was attained in 20 (80%) of 25 complete responders and 28 (57%) of 49 patients overall. 13 responders ceased all therapy; among these all 11 minimal residual disease-negative responders remain progression-free off therapy. Two with minimal residual disease-positive complete response progressed after 24 months off therapy and re-attained response after re-initiation of venetoclax. A substantial proportion of patients achieved an overall response with the combination of venetoclax and rituximab including 25 (51%) of 49 patients who achieved a complete response and 28 (57%) of 49 patients who achieved negative marrow minimal residual disease with acceptable safety. The depth and durability of responses observed with the combination offers an attractive potential treatment option for patients with relapsed or refractory chronic lymphocytic leukaemia and could allow some patients to maintain response after discontinuing therapy, a strategy that warrants further investigation in randomised studies. AbbVie Inc and Genentech Inc.

Combined targeting of BCL-2 and BCR-ABL tyrosine kinase eradicates chronic myeloid leukemia stem cells.

BCR-ABL tyrosine kinase inhibitors (TKIs) are effective against chronic myeloid leukemia (CML), but they rarely eliminate CML stem cells. Disease relapse is common upon therapy cessation, even in patients with complete molecular responses. Furthermore, once CML progresses to blast crisis (BC), treatment outcomes are dismal. We hypothesized that concomitant targeting of BCL-2 and BCR-ABL tyrosine kinase could overcome these limitations. We demonstrate increased BCL-2 expression at the protein level in bone marrow cells, particularly in Lin(-)Sca-1(+)cKit(+) cells of inducible CML in mice, as determined by CyTOF mass cytometry. Further, selective inhibition of BCL-2, aided by TKI-mediated MCL-1 and BCL-XL inhibition, markedly decreased leukemic Lin(-)Sca-1(+)cKit(+) cell numbers and long-term stem cell frequency and prolonged survival in a murine CML model. Additionally, this combination effectively eradicated CD34(+)CD38(-), CD34(+)CD38(+), and quiescent stem/progenitor CD34(+) cells from BC CML patient samples. Our results suggest that BCL-2 is a key survival factor for CML stem/progenitor cells and that combined inhibition of BCL-2 and BCR-ABL tyrosine kinase has the potential to significantly improve depth of response and cure rates of chronic-phase and BC CML.