Upregulation Of Mcl-1 Research Articles

Pre-Clinical Development of Targeted Therapies for Double Hit (DH) Diffuse Large B-Cell Lymphoma (DLBCL)

Background: Gene expression profile classifies DLBCL into three subtypes with distinct pathogenesis and clinical outcomes: activated B-cell (ABC), germinal center B-cell (GCB) and primary mediastinal B-cell lymphoma (PMBL). DH-DLBCL is a distinct subgroup, with a poor clinical outcome, harboring concurrent gene translocations involving the C-MYC and either/both BCL2 or BCL6 proto-oncogenes. DH-DLBCL has inferior response rates to rituximab-based chemotherapy, and a shorter progression-free survival (PFS)/overall survival (OS). In addition, pts with DH-DLBCL who are refractory to frontline chemoimmunotherapy fare dismally; a retrospective review at our institute yielded survival rates of zero and median OS of 6 months in these patients, highlighting the need for novel therapies. DH-DLBCL is characterized by de-regulation of apoptosis and cell cycle progression, leading to rapid proliferation of neoplastic cells, and resistance to apoptotic stimuli. Upregulation of the anti-apoptotic protein Mcl-1 is implicated in chemotherapy resistance and poor clinical outcomes in ABC-DLBCL. Transcriptional regulation of MCL1 by c-MYC might further potentiate Mcl-1 induced resistance to chemotherapy in DH-DLBCL. Specific inhibitors targeting cell pro-survival proteins might provide a novel and alternative therapeutic approach in rituximab-chemotherapy resistant DH-DLBCL. We hypothesize that dual inhibition of anti-apoptotic proteins Bcl-2 and Mcl-1 using readily available agents (Venetoclax and Proteasome inhibitors) is an effective strategy in inducing lymphoma cell death. Materials and Methods: We conducted pre-clinical in vitro and in vivo models targeting Bcl-2 with Venetoclax and Mcl-1 with either a c-Myc inhibitor (JQ-1) or Proteasome inhibitor (Carfilzomib) in DHL. Val, DOHH-2, and ROS-50 cells were exposed to Venetoclax (0-10 uM), JQ-1 (0-100 uM) and Carfilzomib (CFZ) (0-50 nM) for 24, 48 and 72 hours. IC50 drug concentrations were calculated for each agent. Subsequently, DHL cells were exposed to Venetoclax in combination with JQ-1 or CFZ for 48 hours. Cell viability was determined by Cell Titerglo. Coefficient of synergy was calculated using CalcuSyn. In addition, induction of apoptosis was evaluated by flow cytometry (Annexin V/PI staining) and changes in Bcl-2 family members and Caspase activation were determined by Western blotting. Primary tumor cells isolated from B-cell lymphoma patients (N= 15) including DHL were exposed ex vivo to Venetoclax +/- Carfilzomib. For in vivo experiments 6-8 weeks old severe combined immunodeficiency (SCID) mice were inoculated with 10x106 ROS-50 cells via tail vein injection (IV). Subsequently, animals were treated with Venetoclax (100mg/kg/dose via gastric lavage on days 3-7 and 10-14); Carfilzomib (2mg/kg/dose IV days 3, 4, 10, 11), or both agents. A group of untreated animals was used as a control. Differences in survival were evaluated between treatment groups. Results: In vitro, Venetoclax, JQ-1, and CFZ induced cell death in a dose- and time-dependent manner. Significant synergistic activity was observed by combining both Venetoclax and CFZ and to a lesser degree with JQ-1. Similar findings were observed in primary tumor cells isolated from B-cell lymphoma patients. Annexin/PI staining with the combination of Venetoclax and CFZ resulted in caspase dependent apoptosis. Western blotting confirmed changes in Bcl-2 family members favoring an apoptotic state as well as PARP cleavage following in vitro exposure to Venetoclax and CFZ. In vivo the combination of Venetoclax with Carfilzomib resulted in significant increased survival increase (P<0.05) when compared to single agent Venetoclax or CFZ. Conclusion: Our data suggests that Venetoclax exhibits strong synergistic activity with Carfilzomib in vitro and in vivo . Perhaps related to the synergy observed between Venetoclax and CFZ, is the effects of proteasome inhibition on Mcl-1 levels. Combination therapy targeting the proteasome inhibition and BCL-2 may provide an alternative therapeutic approach in DH-DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Combinatorial Treatment of Chronic Lymphocytic Leukemia with Ibrutinib and Venetoclax Is Superior to Treatment with Single Agents in the TCL1 Mouse Model

IntroductionTargeted therapy with the BTK inhibitor ibrutinib or the BH3-mimetic venetoclax has revolutionized the treatment of chronic lymphocytic leukemia (CLL). These inhibitors have a distinct mechanism of action: ibrutinib impacts BCR signaling through BTK inhibition, resulting in displacement of CLL cells from the protective lymph node environment and inhibition of proliferation whereas venetoclax directly kills CLL cells directly by inhibiting anti-apoptotic BCL-2.While both ibrutinib and venetoclax are effective treatments for CLL as single agents, it may be beneficial to combine these treatments. There are indications that cell killing by venetoclax may not be as effective in the lymph node micro-environment due to local upregulation of BCL-XL and MCL-1 [Thijssen et al. Haematologica 2015]. Ibrutinib displaces CLL cells to the blood but does not kill them. Neither agent is curative and both are taken until resistance/relapse occurs, or until treatment is interrupted due to toxicities. Although clinical trials combining these 2 agents are currently underway, their synergistic effects are still largely hypothetical. Therefore we sought to investigate the merits of combining venetoclax with ibrutinib in the murine TCL1 transgenic model for CLL.MethodsIn the present study, immune competent C57BL/6J mice received an adoptive transfer (AT) of TCL1 splenocytes, obtained from full leukemic TCL1 transgenic mice with the same background. Three weeks after AT, when CD5/CD19% reached >90% of all B cells, mice were treated for 4 weeks with venetoclax, ibrutinib or a combination of ibrutinib and venetoclax. Ibrutinib was provided in the drinking water at a concentration of 0.16 mg/ml. Venetoclax was administered daily intra-gastric, at a dose of 100 mg/kg. CLL development was followed by flow cytometry measurements in the peripheral blood.ResultsWithin the first 5 days of treatment, mice receiving ibrutinib single agent showed a rapid transient lymphocytosis (mean WBC numbers at start of treatment: 2.03*107/mL, peak mean at day 2 of treatment: 1.2*108/mL), followed by a reduction in white blood cell (WBC) numbers. Control mice experienced a rapid progression of disease, reaching a mean WBC count of 2.18*108/mL when they had to be sacrificed due to overt leukemia, 8 weeks following AT. Venetoclax and the combination treatment resulted in rapid reductions in WBC count (Figure 1). The maximum drop of WBC counts in the 3 treatment arms as compared to baseline were as follows: ibrutinib 50% at week 6, venetoclax 70% at week 4, combination: 91% at week 5, p<0.001). The mice receiving either monotherapy showed signs of relapse after 4 weeks of treatment, which was manifested by an increase in WBC counts, whereas the combination treatment did not relapse in this timeframe.Spleen sizes of all mice at end of treatment were compared with control mice at week 5. Spleen sizes were significantly reduced in both monotherapies (% reduction: ibrutinib 47%, venetoclax 44%) and even further reduced in mice that received combination therapy (% reduction: 84%) (Figure 2). Untreated mice displayed a disturbed splenic architecture with massively enlarged marginal zones and a loss of demarcation between B and T cells, which was partially restored only in combination treatment.The percentage of infiltrating CLL cells in the spleen was not significantly altered in the monotherapies (67% and 66% of ibrutinib and venetoclax respectively), but was significantly lower in combination treated mice (45%, p<0.001). Number of proliferating splenocytes was only reduced in mice treated with ibrutinib (28%), while cleaved caspase-3 positive B lymphocytes levels, a marker for apoptosis, was only increased in mice treated with venetoclax.Ongoing experiments are aimed at determining levels of BCL-2 and BH3-only members, and functionality of BCR signaling in correlation with the observed effects. Furthermore, we are currently confirming whether the combination results in sustained response compared to the single treatments.ConclusionsIn conclusion, these first data from the TCL1 CLL model support the combination treatment of venetoclax and ibrutinib. Even in this short treatment timeframe, there is a more durable response with the combination treatment, which may be due to the elimination of reservoirs of CLL cells where resistance to single agent treatment can develop. [Display omitted] DisclosuresSlinger:Johnson & Johnson: Research Funding; Abbvie: Research Funding. Balasubramanian:Janssen Research & Development: Employment, Equity Ownership. Eldering:Roche: Research Funding; Gilead: Research Funding; Celgene: Research Funding. Kater:Johnson & Johnson: Research Funding; Abbvie: Research Funding; Celgene: Consultancy, Research Funding.

Targeting Mcl-1 By Proteasome Inhibitors Enhances the Anti-Tumor Activity of Venetoclax in Mantle Cell Lymphoma Pre-Clinical Models

Background: Mantle cell lymphoma (MCL) accounts for 6% of all non-Hodgkin lymphomas and is a therapeutic challenge. At the molecular level, MCL is characterized by the deregulation of Bcl-2 family members (Mcl-1, BIM) altering apoptosis, as well as, cell cycle (cyclin D1) partially regulated by the ubiquitin-proteasome system (UPS). Inhibitors of the UPS, also known as proteasome inhibitors (PI), have been tested against MCL. The lethality by PIs has been associated with pronounced up-regulation of the pro-apoptotic protein Noxa, which prevented the upregulation of Mcl-1. Changes in the ratio of Mcl-1 and Bak/Bax result in the release of Bak triggering apoptosis particularly in cells with functional p53. Venetoclax is a highly selective Bcl-2 inhibitor, BH-3 mimetic with potent activity against MCL, follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) cell lines, as well as in a t(14;18)-carrying xenograft model with an established tolerability and safety in a recent phase I trial. We sought to look at further enhancing the pre-clinical activity of venetoclax using PIs in MCL models. We hypothesized that dual inhibition of anti-apoptotic proteins Bcl-2 and Mcl-1 could result in synergistic activity that could be translated clinically. Materials and Methods: We performed in vitro studies on a panel cytarabine sensitive (Rec-1, Jeko, Granta, HBL-2, Z-138 and Mino) and resistant (araC) cell line clones (Jeko araC, HBL-2 araC, and Mino araC). MCL cell lines were exposed to venetoclax, carfilzomib, ixazomib and bortezomib for 24, 48 and 72 hours. Cell viability was calculated measuring the ATP content. IC50 drug concentrations were calculated for each agent. Subsequently, MCL cell lines were exposed to escalating doses of venetoclax (0.001uM-5uM) and carfilzomib, an irreversible PI (1.5625nM-50nM); or two clinically available PI: bortezomib (3.125nM-100nM) or ixazomib (3.125nM-100nM). Cell viability was determined by Cell Titerglo. Coefficient of synergy were calculated using CalcuSyn. Primary tumor cells isolated from B-cell lymphoma patients (N= 15) including MCL were exposed ex vivo to venetoclax +/- carfilzomib or bortezomib. Results: In vitro exposure of MCL cell lines to either venetoclax, carfilzomib, bortezomib and ixazomib induced cell death in a dose- and time-dependent manner. Anti-tumor activity was observed in cytarabine sensitive or resistant cell lines. Significant synergistic activity was observed by combining both venetoclax with carfilzomib or ixazomib at known sub-therapeutic and therapeutic doses of individual agents. Similar findings were observed in primary tumor cells isolated from B-cell lymphoma patients. Conclusion: Our data suggests that venetoclax exhibits strong synergistic activity with carfilzomib and ixazomib in vitro and ex vivo MCL models. This pharmacological interaction could be related to the effects of proteasome inhibition on Mcl-1 in MCL cells, further enhancing the activity of venetoclax. In vivo experiments will further confirm the synergistic activity observed between BH3 mimetics and PIs. (Supported by a NCI SPORE Lymphoma grant: 5 P50 CA130805-04, an NIH grant R01 CA136907-01A1 and a grant from The Roswell Park Cancer Institute Alliance Foundation) DisclosuresNo relevant conflicts of interest to declare.

Venetoclax As a Single Agent and in Combination with PI3K-mTOR1/2 Kinase Inhibitors Against Ibrutinib Sensitive and Resistant Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma and prognosis remains poor in MCL patients due to minimal residual disease (MRD), subsequent emergence of drug resistance and lymphoma progression. Ibrutinib is a bruton's tyrosine kinase (BTK) inhibitor that has high response rates in patients with MCL, however, over 30% of MCL patients showed partial or complete lack of response to ibrutinib and experienced disease progression within 12 months of treatment. Alarmingly, once patients relapse after ibrutinib treatment, the 1-year survival rate is only 22%. Our study demonstrated that the major hallmarks of ibrutinib resistant (IR) MCL are increased mitochondrial apoptosis priming, increased BCL-2 dependence, and sustained PI3K-AKT-mTOR pathway activation accompanied by enhanced stromal cell interaction. Sustained BCL-2 and PI3K-AKT-mTOR pathways cooperatively dictate interaction (adhesion) between MCL and stroma, promote MCL IR and proliferation. Disruption of both pathways establishes a novel ‘triple-hit’ or ‘multi-hit’ strategy: targeting the pathway (BCL-2) related to survival, targeting the pathway (PI3K-mTOR) related to cell proliferation, culminating in disruption of the lymphoma-stroma adhesion/interaction to sensitize and enhance cytotoxicity. BEZ235 and AZD8055 synergized with ABT-199 to suppress MCL viability and attenuated clonogenic growth in both ibrutinib sensitive and resistant MCL lines and primary cells. Moreover, we observed that compensatory upregulation of MCL1 following ABT-199 exposure could be abrogated by BEZ235 and AZD8055 - both in ibrutinib sensitive and IR cells. The combination of BEZ235 and AZD8055 with ABT-199 triggered a more dramatic reduction of MCL-1 protein expression, accompanied by an increase in apoptotic PARP cleavage, both in IR MCL cell lines and primary MCL. Together, these data suggest a novel strategy to overcome the dismal prognosis associated with IR. DisclosuresNo relevant conflicts of interest to declare.

Immunostimulatory CpG Oligodeoxynucleotides Induce Sustained ERK2 Activation and Strong Proliferative Responses in CLL B-Cells from Patients with Progressive Disease.

Immunostimulatory DNA sequences that are present in microbial DNA, DNA-containing immune complexes and synthetic CpG-oligodeoxynucleotides (ODNs) can activate cells of the immune system via Toll-like receptor 9 (TLR9). Normal B-cells respond to such stimulation by upregulation of co-stimulatory molecules, proliferation and prolonged survival. The response of chronic lymphocytic leukemia B-cells to CpG ODN stimulation appears to be more variable, since both proliferation and induction of apoptosis have been reported. Because CLL B-cells with mutated and unmutated Ig VH genes show distinct clinical and biological behaviors, we compared their responsiveness to CpG ODN stimulation in terms of proliferation, survival, activation of TLR9 signaling pathways and expression of cell-cycle regulatory and anti-apoptotic proteins. A significant proliferative response was induced in most of the VH unmutated CLL samples (14/23), whereas 3H thymidine incorporation was low or absent in the majority of the VH mutated cases (22/27, P = 0.005). More strikingly, stratification of the CLL cases based on the clinical course showed strong proliferative responses in 14/21 cases with progressive disease and in only 5/29 cases with stable disease (P=0.001). The absence of a proliferative response to CpG ODN stimulation in the stable/VH mutated cases was not caused by different early signaling events, since phosphorylation of JNK, ERK1/2 and Akt, as well as degradation of IkB, was detected in almost all investigated samples (7/7 proliferating and 5/6 nonproliferating). However, a notable difference was observed in the duration of ERK2 activation, which was transient in the nonproliferating CLL B-cells, but lasted more than 24 hours in the proliferating cases. This difference was associated with dimished phosphorylation of the retinoblastoma protein (pRb) and less pronounced or absent induction of cyclin E and cyclin A in the nonproliferating cases, suggesting a block in the G1/S phase transition. On the other hand, cyclin D3 was induced and p27KIP1 was downregulated in all samples, indicating that CLL B-cells from both subgroups can enter the G1 phase of the cell cycle. Analysis of CLL B-cell survival by Annexin V/PI staining and PARP cleavage after 48 hours stimulation with CpG ODN showed significant levels of apoptosis in most of the nonproliferating cases. In contrast, proliferating CLL B-cells remained viable after CpG ODN stimulation, which was associated with upregulation of the anti-apoptotic proteins Mcl-1 and Bcl-xL. These data indicate that immunostimulatory CpG oligodeoxynucleotides can induce distinct responses in progressive/VH unmutated and stable/VH mutated CLL B-cells, which are characterized by proliferation and inhibition of spontaneous apoptosis in the former and induction of apoptosis in the latter group of cases. Although both subgroups show similar early activation of TLR9 signaling pathways and both enter into the G1 phase of the cell cycle, the absence of sustained ERK2 activation precludes cell cycle progression in most stable/VH mutated CLL B-cells. The greater capacity to traverse the cell cycle and proliferate in response to immunostimulatory CpG DNA may therefore be related to the more aggressive clinical course that is associated with CLL B-cells that express unmutated VH genes.

Identification of the Core MicroRNAs and Potential Molecular Mechanismsin Sarcoidosis Using Bioinformatics Analysis.

Sarcoidosis is a systemic heterogeneous inflammatory disease; however, the etiology and pathogenesis of sarcoidosis are still unknown. Herein, we investigated the core microRNAs and potential molecular mechanisms in sarcoidosis. The DE-miRNAs were diagnosed using the LIMMA software package. DIANA-mirPath was employed to perform pathway and GO enrichment analysis of the DE-miRNAs. PPI networks and miRNA-target gene regulatory networks were used to obtain insight into the actions of DE-miRNAs. Expression of the hub genes along with miRNAs was validated in clinical specimens. Overall, 266 DE-miRNAs were screened. Among these DE-miRNAs, hsa-miR-144, hsa-miR-126, as well as hsa-miR-106a were the upmost upregulated miRNAs; hsa-miR-151-3p, hsa-miR-320d, and hsa-miR-324-3p were the top downregulated miRNAs. NR3C1, ZBTB7A, NUFIP2, BZW1, ERGIC2, and VEGFA were mapped as the most targeted hub genes in the upregulation of miRNAs, and MCL1 and SAE1 were the most targeted hub genes in the downregulation of miRNA. VEGFA and NR3C1 were selected and potentially modulated by hsa-miR-20b, hsa-miR-126, and hsa-miR-106a. In sarcoidosis pathological tissue, hsa-miR-126 was highly expressed, and VEGFA and NR3C1 were overexpressed. In conclusion, our results revealed the dysregulation of hsa-miR-126 and a potential regulatory mechanism for pathogenesis in sarcoidosis.

Bone Marrow Mesenchymal Stromal Cells Can Render Multiple Myeloma Cells Resistant to Cytotoxic Machinery of CAR T Cells through Inhibition of Apoptosis.

The microenvironment of multiple myeloma (MM) can critically impair therapy outcome, including immunotherapies. In this context, we have earlier demonstrated that bone marrow mesenchymal stromal cells (BMMSC) protect MM cells against the lytic machinery of MM-reactive cytotoxic T cells (CTL) and daratumumab-redirected natural killer (NK) cells through the upregulation of antiapoptotic proteins Survivin and Mcl-1 in MM cells. Here, we investigated the significance of this mode of immune escape on T cells engineered to express chimeric antigen receptors (CAR T cells). We tested the cytolytic ability of a panel of 10 BCMA-, CD38-, and CD138-specific CAR T cells with different affinities against a model MM cell line and against patient-derived MM cells in the presence versus absence of BMMSCs. Although BMMSCs hardly protected MM cells from lysis by high-affinity, strongly lytic BCMA- and CD38-CAR T cells, they significantly protected against lower affinity, moderately lytic BCMA-, CD38-, and CD138-specific CAR T cells in a cell-cell contact-dependent manner. Overall, there was a remarkable inverse correlation between the protective ability of BMMSCs and the lytic activity of all CAR T cells, which was dependent on CAR affinity and type of costimulation. Furthermore, BMMSC-mediated resistance against CAR T cells was effectively modulated by FL118, an inhibitor of antiapoptotic proteins Survivin, Mcl-1, and XIAP. These results extend our findings on the negative impact of the microenvironment against immunotherapies and suggest that outcome of CAR T cell or conventional CTL therapies could benefit from inhibition of antiapoptotic proteins upregulated in MM cells through BMMSC interactions.

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.

ANO1 Expression Orchestrates p27Kip1/MCL1-Mediated Signaling in Head and Neck Squamous Cell Carcinoma.

Simple SummaryOur aim was to elucidate the molecular mechanisms of how ANO1 contributes to oncogenic processes in squamous cell carcinoma of the head and neck (HNSCC). We explored transcriptional programs influenced by ANO1 knockdown in patient-derived UT-SCC cell lines with 11q13 amplification and ANO1 overexpression. ANO1 depletion led to downregulation of broad pro-survival BCL2 family protein members, including MCL1, and simultaneously induced upregulation of the cell cycle inhibitor p27Kip1 and its redistribution from the cytoplasm into the nucleus in the studied HNSCC cells. Gene set enrichment analysis highlighted pathways associated with perturbed cell cycle and apoptosis in the ANO1-depleted samples. Silencing of ANO1 and application of an ANO1-targeting small-molecule inhibitor led to ANO1 degradation and reduction of cell viability. These findings suggest that ANO1 has drug target potential that deserves further evaluation in preclinical in vivo models.Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors that derive from the mucosal epithelium of the upper aerodigestive tract and present high mortality rate. Lack of efficient targeted-therapies and biomarkers towards patients’ stratification are caveats in the disease treatment. Anoctamin 1 (ANO1) gene is amplified in 30% of HNSCC cases. Evidence suggests involvement of ANO1 in proliferation, migration, and evasion of apoptosis; however, the exact mechanisms remain elusive. Aim of this study was to unravel the ANO1-dependent transcriptional programs and expand the existing knowledge of ANO1 contribution to oncogenesis and drug response in HNSCC. We cultured two HNSCC cell lines established from primary tumors harboring amplification and high expression of ANO1 in three-dimensional collagen. Differential expression analysis of ANO1-depleted HNSCC cells demonstrated downregulation of MCL1 and simultaneous upregulation of p27Kip1 expression. Suppressing ANO1 expression led to redistribution of p27Kip1 from the cytoplasm to the nucleus and associated with a cell cycle arrested phenotype. ANO1 silencing or pharmacological inhibition resulted in reduction of cell viability and ANO1 protein levels, as well as suppression of pro-survival BCL2 family proteins. Collectively, these data provide insights of ANO1 involvement in HNSCC carcinogenesis and support the rationale that ANO1 is an actionable drug target.

Aggressive B-cell Lymphoma with MYC/TP53 Dual Alterations Displays Distinct Clinicopathobiological Features and Response to Novel Targeted Agents.

Diffuse large B-cell lymphoma (DLBCL) is the major type of aggressive B-cell lymphoma. High-grade B-cell lymphoma (HGBCL) with MYC/BCL2 double-hit (DH) represents a distinct entity with dismal prognosis after standard immunochemotherapy in the current WHO lymphoma classification. However, whether TP53 mutation synergizes with MYC abnormalities (MYC rearrangement and/or Myc protein overexpression) contributing to HGBCL-like biology and prognosis is not well investigated. In this study, patients with DLBCL with MYC/TP53 abnormalities demonstrated poor clinical outcome, high-grade morphology, and distinct gene expression signatures. To identify more effective therapies for this distinctive DLBCL subset, novel MYC/TP53/BCL-2-targeted agents were investigated in DLBCL cells with MYC/TP53 dual alterations or HGBCL-MYC/BCL2-DH. A BET inhibitor INCB057643 effectively inhibited cell viability and induced apoptosis in DLBCL/HGBCL cells regardless of MYC/BCL2/TP53 status. Combining INCB057643 with a MDM2-p53 inhibitor DS3032b significantly enhanced the cytotoxic effects in HGBCL-DH without TP53 mutation, while combining with the BCL-2 inhibitor venetoclax displayed potent therapeutic synergy in DLBCL/HGBCL cells with and without concurrent TP53 mutation. Reverse-phase protein arrays revealed the synergistic molecular actions by INCB057643, DS3032b and venetoclax to induce cell-cycle arrest and apoptosis and to inhibit AKT/MEK/ERK/mTOR pathways, as well as potential drug resistance mechanisms mediated by upregulation of Mcl-1 and RAS/RAF/MEK/ERK pathways. In summary, these findings support subclassification of DLBCL/HGBCL with dual MYC/TP53 alterations, which demonstrates distinct pathobiologic features and dismal survival with standard therapy, therefore requiring additional targeted therapies. IMPLICATIONS: The clinical and pharmacologic studies suggest recognizing DLBCL with concomitant TP53 mutation and MYC abnormalities as a distinctive entity necessary for precision oncology practice. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/2/249/F1.large.jpg.

The Anti-apoptotic Role of 3'-Untranslational Region in Response to Angiotensin II via Mcl1 Expression.

Myeloid cell leukemia 1 (Mcl1), an abundant protein in the myocardium, plays an essential role in fibrosis and anti-inflammation in cardiomyocytes to prevent heart failure. However, whether Mcl1 3′-untranslated regions (3′-UTR) has the cardio-protecting function remains unclear. Down-regulation of Mcl1 was observed in adult mice heart tissues after Angiotensin II (Ang II) treatment. Consistent with in vivo results, the reduction of Mcl1 expression was identified in Ang II-treated neonatal cardiomyocytes. Mechanistically, Mcl1 3′-UTR prevented Ang II-induced cardiac apoptosis via up-regulation of Mcl1 and an angiogenic factor with a G-patch domain and a forkhead-associated domain 1 (Aggf1), which plays cardiac-protective role. Our work broadens the scope of gene therapy targets and provides a new insight into gene therapy strategies involving mRNAs’ 3′-UTRs application.

A Randomized Phase 2 Study of Pevonedistat, Venetoclax, and Azacitidine Versus Venetoclax Plus Azacitidine in Adults with Newly Diagnosed Acute Myeloid Leukemia (AML) Who Are Unfit for Intensive Chemotherapy

A Randomized Phase 2 Study of Pevonedistat, Venetoclax, and Azacitidine Versus Venetoclax Plus Azacitidine in Adults with Newly Diagnosed Acute Myeloid Leukemia (AML) Who Are Unfit for Intensive Chemotherapy

Revealing Transcriptome Deregulation upon Genomic Complexity in Multiple Myeloma

Introduction Multiple Myeloma (MM) is characterized by hyperdiploidy (HD) or immunoglobulin gene (IgH) rearrangements as initiating events. Clonal heterogeneity is a hallmark of its biology as highlighted by Next Generation Sequencing. In this context, data on the impact of peculiar mutations, copy number aberrations (CNAs), and chromosomal rearrangements (CRs) at the transcriptomic level are still scanty. In this study, we aimed to dissect the transcriptional deregulation promoted by the most recurrent genomic drivers. Based on this geno-trascriptomic link, we also aimed to identify biomarkers that could suggest personalized treatments. Methods We analyzed 517 newly diagnosed patients from the IA12 release of the CoMMpass study, focusing on mutations in MM driver genes, structural variants, copy number segments and raw transcript counts. RNAseq data was processed with the VOOM/LIMMA pipeline. To perform an in-silico drug sensitivity screen, we anchored cell lines to patients samples using the Celligner algorithm and interrogated the DepMap dataset. Results We first analyzed the global impact of genetic aberrations on the transcriptome. Chr(1q)amp/gain, followed by IgH translocations and HD showed the highest number of deregulated transcripts. Individual mutations had much less impact, with the exception of NRAS and chr(13q) genes (DIS3, TGDS, RB1). Next, we investigated differential influence between hotspots (HS) vs nonHS mutations within driver genes. KRAS and NRAS, showed little changes between nonHS and wild type (WT), as the transcriptome was mostly impacted by HS mutations. IRF4 K123 showed a specific transcriptional profile, while nonHS mutations still carried functional relevance although on different genes. For BRAF, the kinase dead D594 mutation surprisingly impacted the most in comparison to V600 and WT cases. Next, we explored the effect of bi-allelic genetic events with known prognostic impact. TP53 double-hits were associated with an upregulation of PHF19, a MM poor prognostic marker, and downregulation of SLAMF7, a new immunotherapy target. CYLD and TRAF3 double-hits correlated with NF-κB pathway activation, and the former showed a significant BCL2 upregulation. Bi-allelic events on chr13 exhibited gene-specific consequences: DIS3 inactivation deregulated mostly lncRNAs, while TGDS impacted on genes involved in cell-cycle regulation. Regarding chromosomal gains, only chr(1q)amp (&amp;gt; 3 copies) showed a gene dosage effect with upregulation of the potential therapeutic targets MCL1 and SLAMF7. Given that the BCL2 axis was perturbated by several genetic alterations, we systematically compared the expression levels of BCL2, NOXA, MCL1 and BCL2L1 in CYLD inactivated, t(11;14) and chr(1q)amp patients. BCL2 levels were higher in the CYLD group, which parallels with the overexpression of the anti-apoptotic gene BCL2L1. NOXA, which promotes MCL1 degradation, was significantly upregulated in t(11;14). Chr(1q)amp patients showed a concomitant MCL1 overexpression and NOXA downregulation. To correlate these results to drug sensitivity, we performed an in-silico screen. We first selected MM and lymphoma cell lines from the DepMap dataset based on a gene expression profile that was most similar to the MM samples, then analyzed candidate drugs. The SKMM2 MM cell line, harboring t(11;14), del(CYLD) e NOXAamp was highly sensitive to Venetoclax. The same was true for the lymphoma ones RI1 and OCI-LY3, both harboring NOXAamp, but negative for t(11;14). On the contrary, the U266 and MOLP8 both with t(11;14) carrying a MCL1amp due to a chr(1q)amp were fully resistant. Of note, these latter resulted sensitive to the pan-BCL2 axis inhibitor Sabutoclax. Conclusions Our study suggests a link between the genomic architecture and transcriptome in MM, where CNAs and CRs had a stronger impact on expression than gene mutations. However, given that not all mutations are identical, HS ones need further testing as they may represent a future treatment target. Moreover, the mutational status is crucial since, while mono-allelic events are often of little transcriptional value, compound heterozygosity carries a huge influence on transcriptomic which provides biological basis for the observed prognostic impact of "double-hit" MM. Finally, we suggest that a comprehensive profiling of the BCL2 pathway may identify biomarkers of sensitivity to BCL2 inhibitors in addition to the t(11;14). Disclosures D'Agostino: GSK: Membership on an entity's Board of Directors or advisory committees. Corradini:Celgene: Consultancy, Honoraria, Other: Travel and accommodations paid by for; Sanofi: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Other: Travel and accommodations paid by for; Incyte: Consultancy; Daiichi Sankyo: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Other; BMS: Other; F. Hoffman-La Roche Ltd: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: Travel and accommodations paid by for; Novartis: Consultancy, Honoraria, Other: Travel and accommodations paid by for; Servier: Consultancy, Honoraria; Kite: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Other: Travel and accommodations paid by for; KiowaKirin: Consultancy, Honoraria. Bolli:Celgene: Honoraria; Janssen: Honoraria.

Targeting 1q21 Amplification with APG2575 and Lenalidomide to Sensitize BCL-2 Inhibition with the Decrease of MCL-1 Protein in High Risk MM Models

Background Multiple myeloma (MM) is a heterogenous plasma cell malignancy. About 30-40% of newly diagnosed and 20-60% of relapsed/refractory MM patients carry 1q21 amplification worldwide, a high-risk indicator for poor prognosis in RRMM. Different BCL-2 family anti-death proteins play important roles in MM survival and drug resistance. High expression of BCL-2 due to t (11;14) renders cell vulnerability to BCL-2 antagonist, however, patients carrying other genetic abnormality including 1q21 amplification have limited response to the newly emerged treatment choice. With MCL-1 upregulation accompanied with 1q21 amplification, we tested whether BCL-2 antagonist combined with IMiDs (immunomodulatory imide drugs), improves cell killing in high-risk MM patient models, including those resistant to bortezomib and lenalidomide. For that aim, we studied APG-2575, a novel and potent BCL-2 inhibitor developed by Ascentage Pharma Group and it is currently in clinical trials for hematologic malignances, including MM. Methods 1. Cells were treated with APG-2575 single agent or in combination with lenalidomide; 2. Cell line viability was assessed by CellTiter-Glo (CTG) assay; 3. Flow cytometry analysis was used to detect CD138+ cell surface marker in primary cells derived from MM patients; 4. Western blot analysis for BCL-2 family and IMiD signaling proteins. Results We first determined the cell sensitivity of APG-2575 as a single agent in a panel of MM cell lines, and as expected, those carrying t (11;14) were very sensitive to APG-2575, with low IC50 values ranging 7-23 nM. The IC50 values for cells carrying other genetic markers were greater than 5-10 μM. We then evaluated cell-death inducing activity of APG-2575 in MM patient-derived primary cells ex vivo, which were freshly prepared from patients' bone marrow aspirates. Primary cells were treated with APG-2575 or ABT-199 (venetoclax) for 18-24 hours, and the loss of CD138 surface marker was used to quantify cell death. As shown in Figure 1a, APG-2575 induced cell death (CD138+ loss) in a dose-dependent manner, and significant cell death was observed at 0.37-3.3 μM of APG-2575, indicating primary cells more sensitive than MM cell lines. Interestingly, the sensitivity to APG-2575 is similar in primary MM cells with or without 1q21 amplification. Since moderate cell death inducing activity was observed in MM cells when APG-2575 used as a single agent, we combined APG-2575 with lenalidomide. Cell death was increased in the combination groups compared with single agent, and it was dose-dependent (Figure 1b).Similar enhanced antiproliferative activity was confirmed in RPMI 8226 cell line, which carries 1q21 amplification (Figure 1c). We furthered to understand the mechanism of action (MoA) of this combination. In Figure 1d, Western blot analysis of RPMI 8226 cell line revealed that IKZF1 and IKZF3 proteins from the NF-KB pathway were downregulated by lenalidomide. And the decrease of MCL-1 protein and the strong induction of pro-death protein BAK were evident in the combination group of APG-2575 and lenalidomide, which helps to illustrate MoA underlying the synergistic effect of APG-2575 and lenalidomide in MM models. Conclusions In both cell lines and primary samples derived from MM patients, APG-2575 demonstrates cell death inducing activity as a single agent, and enhanced/synergistic effects when it combines with lenalidomide in RRMM resistant to lenalidomide and bortezomib. The combination decreases IKZF1, IKZF3 and MCL-1 proteins, and upregulates pro-death protein BAK, thus providing a strong rationale to combine BCL-2 inhibitor and lenalidomide to treat high-risk patient populations carrying 1q21 amplification. Disclosures FU: Ascentage Pharma (SuZhou) Co., Ltd: Other: research agreement contract . Yin:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment. Mao:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment. Deng:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment. Fang:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment. Yang:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests. Zhai:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests.

Interim Results of the Phase I/II Study of the Ponatinib, Venetoclax and Dexamethasone for Patients with Relapsed or Refractory Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

Interim Results of the Phase I/II Study of the Ponatinib, Venetoclax and Dexamethasone for Patients with Relapsed or Refractory Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

Combinations of proteasome inhibitors with obatoclax are effective for small cell lung cancer.

Proteasome inhibitors, bortezomib (BTZ), and carfilzomib (CFZ) are approved drugs for hematological malignancies, but lack anticancer activities against most solid tumors. Small cell lung cancer (SCLC) is a very aggressive neuroendocrine carcinoma of the lungs demanding effective therapy. In this study we investigated whether BTZ or CFZ combined with obatoclax (OBX), an antagonist for MCL-1 and a pan-BCL family inhibitor, could cause synergistic growth inhibition of SCLC cells. We showed that combined application of BTZ or CFZ with OBX caused synergistic growth inhibition of human SCLC cell lines (H82, H526, DMS79, H196, H1963, and H69) than single agent alone. Both BTZ-OBX and CFZ-OBX combinations displayed marked synergism on inducing apoptosis (~50% increase vs BTZ or CFZ alone). A comprehensive proteomics analysis revealed that BTZ preferentially induced the expression of MCL-1, an antiapoptotic protein, in SCLC cells. Thus, proteasome inhibitor-OBX combinations could specifically induce massive growth inhibition and apoptosis in SCLC cells. Subsequent proteome-wide profiling analysis of activated transcription factors suggested that BTZ- or CFZ-induced MCL-1 upregulation was transcriptionally driven by FOXM1. In nude mice bearing in SCLC H82 xenografts, both BTZ-OBX, and CFZ-OBX combinations exhibited remarkable antitumor activities against SCLC tumors evidenced by significant reduction of tumor size and the proliferation marker Ki-67 signals in tumor tissues as compared with single agent alone. Thus, proteasome inhibitor-OBX combinations are worth immediate assessments for SCLC in clinical settings.

Targeting the Synthetic Vulnerability of PTEN-Deficient Glioblastoma Cells with MCL1 Inhibitors.

PTEN deletion or mutation occurs in 30% to 60% of patients with glioblastoma (GBM) and is associated with poor prognosis. Efficacious therapy for this subgroup of patients is currently lacking. To identify potential target(s) to selectively suppress PTEN-deficient GBM growth, we performed a three-step synthetic lethal screen on LN18 PTEN wild-type (WT) and knockout (KO) isogeneic GBM cell lines using a library containing 606 target-selective inhibitors. A MCL1 inhibitor UMI-77 identified in the screen exhibited excellent suppression on the proliferation, colony formation, 3D spheroid, and neurosphere formation of PTEN-deficient GBM cells. Mechanistically, loss of PTEN in GBM cells led to upregulation of MCL1 in posttranslational level via inhibition of GSK3β, and consequently confer cells resistance to apoptosis. Pharmacologic inhibition or knockdown of MCL1 blocked this PI3K-GSK3β-MCL1 axis and caused reduction of several antiapoptotic proteins, finally induced massive caspase-3 cleavage and apoptosis. In both subcutaneous and orthotopic GBM models, knockdown of MCL1 significantly impaired the in vivo growth of PTEN-deficient xenografts. Moreover, the combination of UMI-77 and temozolomide synergistically killed PTEN-deficient GBM cells. Collectively, our work identified MCL1 as a promising target for PTEN-deficient GBM. For future clinical investigations, priority should be given to the development of a selective MCL1 inhibitor with efficient brain delivery and minimal in vivo toxicity.

AML-137: Preclinical Evaluation of Cytarabine and Venetoclax in Secondary AML

Context Novel treatment approaches for patients with secondary acute myeloid leukemia (AML) are urgently needed. Secondary AML constitutes about 25-30% of AML and it has been observed that patients with secondary AML have worse responses rates to chemotherapy, increased rates of relapse, and worse overall survival compared to patients with de novo AML. Objective Our long-term goal is to develop more effective therapeutic options for patients with secondary AML. Our objectives in this study are: (i) to demonstrate synergy between cytarabine and venetoclax in secondary AML cell lines and patient-derived samples, (ii) to elucidate the mechanism of this synergy, (iii) functional characterization of Bcl-2 family proteins by BH3 profiling performed on secondary AML cell lines and patient samples. Results Synergy was demonstrated between cytarabine and venetoclax in secondary AML and de novo AML cell lines (Molm13, MV4;11, SKM-1; ML-2) using both an MTT cell viability assay and a flow-cytometry based Annexin-V assay, quantified by the computer software Compusyn. BH3 profiling of these cell lines indicated a dependence on both Bcl-2 and Mcl-1. Interestingly, SKM-1 showed sole dependence on Mcl-1. In SKM-1 cells, western blotting showed increased expression of Mcl-1 after treatment with venetoclax for 8 hrs and decreasing Mcl-1 expression with time (8hrs–72hrs) after treatment with cytarabine. Combination treatment for 24-48 hrs led to Mcl-1 expression that was similar to or less than Mcl-1 expression in control cells. Synergy between cytarabine and venetoclax was confirmed in patient samples. Importantly, BH3 profiling showed that patient samples exhibited dependence on Bcl-2 or both Mcl-1 and Bcl-2. Conclusions There is significant synergy between cytarabine and venetoclax in both de novo and secondary AML cell lines as well as patient samples. One of the potential mechanisms of this synergy is abrogation of Mcl-1 upregulation. Secondary AML exhibits variable functional dependence on Mcl-1 and Bcl-2. These findings support the clinical investigation of cytarabine and venetoclax in patients with secondary AML as well as the use of BH3 profiling as a potential predictive biomarker. Acknowledgements This work was supported by the NIH R01 CA-217141 and AACR Bayer Innovation and Discovery Award to Z. Nikolovska-Coleska.

Abstract 4225: CG-806, a first-in-class FLT3/BTK inhibitor, and venetoclax synergize to inhibit cell proliferation and to induce apoptosis in aggressive B-cell lymphomas

Abstract Double/triple-hit lymphomas (DHL/THL, harboring concurrent MYC, BCL2 and/or BCL6 rearrangements) and double-expressor lymphomas (DEL, with MYC/BCL2 co-overexpression without underlying rearrangements) account for 7-12% and 20-30% of diffuse large B-cell lymphomas (DLBCLs), respectively, and have poor outcome after standard/intensive immunochemotherapy (Petrich, 2014; Smith, 2018). More effective and less toxic treatments for these aggressive B-cell lymphomas represent a significant unmet medical need. Venetoclax, a BCL2 inhibitor, significantly inhibits the proliferation of DLBCL cells in culture but has less promising clinical efficacy as a single agent. CG-806, a FLT3/BTK inhibitor, exhibits potent antitumor activity against cell lines and primary cells over a range of hematologic malignancies by suppressing the driver and compensatory pathways (inhibits phosphorylation of BTK, FLT3, PDGFRα, SYK, SRC, ERK, MAPK, STAT5 and AKT, and decreases MYC level). CG-806 is currently in a Phase I a/b trial in patients with CLL/SLL or non-Hodgkin's lymphomas (NCT03893682). We sought to investigate the antitumor effect of CG-806 alone or in combination with venetoclax on DHL/THL/DEL cells using MTS based proliferation assay, flow cytometry assays of apoptosis and cell cycle, and immunoblotting. In DHL/THL cells VAL, SU-DHL6 and DOHH2 that overexpress MYC and BCL2, CG-806 inhibited their proliferation with IC50s of 0.17, 0.15 and 0.005 µM, respectively. CG-806 was 5-20X more potent than venetoclax and exposure to CG-806 significantly enhanced sensitivity to venetoclax. SU-DHL2, a DHL cell line that does not overexpress MYC or BCL2, was inhibited by CG-806 but not venetoclax (IC50 0.9 vs 6.3 µM, respectively). In the DEL cell line U2933, CG-806 and venetoclax had similar potency as single agents (IC50 1.6 µM); when combined, the IC50 was markedly improved to 0.05 µM. Meanwhile, the combination of CG-806 with venetoclax enhanced induction of apoptosis in DHL/THL/DEL cells in a dose and time-dependent manner as detected by Annexin V positive, cleavage of PARP and Caspase 3. Recent studies ascribed the resistance of DLBCLs to venetoclax to upregulation of MCL1 and MAPK signaling. our study showed that CG-806 inhibited expression of the anti-apoptotic protein MCL1, overcame the effect of venetoclax-induced MCL1 and increased the level of proapoptotic BIM. While venetoclax produced no effect on MAPK signaling, CG-806 alone and in combination with venetoclax reduced p-MEK1/2, p-JNK and MYC in a cell line dependent manner. In conclusion, CG-806 inhibits driver and rescue pathways to directly and potently kill the aggressive B-cell lymphomas including DHL/THL/DEL and enhances the proapoptotic effect of venetoclax, thereby highlighting CG-806 as a promising candidate for the treatment of patients harboring unfavorable BCL2/MYC/BCL6 translocations and/or overexpression and supporting clinical development of CG-806 in patients with B-cell malignancies intolerant, resistant, or refractory to venetoclax. Citation Format: Nasrin Rastgoo, Matthew Thayer, Khalid Benbatoul, Stephen Howell, William Rice, Hongying Zhang. CG-806, a first-in-class FLT3/BTK inhibitor, and venetoclax synergize to inhibit cell proliferation and to induce apoptosis in aggressive B-cell lymphomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4225.

Inhibition of EGFR pathway promotes the cytotoxicity of ABT-263 in human leukemia K562 cells by blocking MCL1 upregulation

ABT-263 induces MCL1 upregulation in cancer cells, which confers resistance to the drug. An increased understanding of the mechanism underlying ABT-263-induced MCL1 expression may provide a strategy to improve its tumor-suppression activity. The present study revealed that ABT-263 reduced the turnover of MCL1 mRNA, thereby upregulating MCL1 expression in human K562 leukemia cells. Furthermore, ABT-263-induced EGFR activation promoted AGO2 phosphorylation at Y393 and reduced miR-125b maturation. Treatment with EGFR inhibitors mitigated MCL1 upregulation induced by ABT-263. Additionally, lithium chloride (LiCl) alleviated ABT-263-induced MCL1 upregulation through EGFR-AGO2 axis-modulated miR-125b suppression. Ectopic expression of dominant negative AGO2(Y393F) or transfection with miR-125b abolished ABT-263-induced upregulation of MCL1 mRNA and protein levels. Co-treatment with either EGFR inhibitors or LiCl collaboratively enhanced ABT-263 cytotoxicity, while MCL1 overexpression eliminated this synergistic effect. Collectively, our data reveal that ABT-263 increases EGFR-mediated AGO2 phosphorylation, which in turn suppresses miR-125b-mediated MCL1 mRNA degradation in K562 cells. The suppression of this signaling pathway results in the synergistic cytotoxic effect of EGFR inhibitors or LiCl and ABT-263.