High Levels Of Mcl-1 Research Articles

Differentiation State Plasticity As a Mechanism of BCL2 Inhibitor Resistance in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is composed of a heterogeneous blast population that exhibits characteristics of normal myeloid cells, including immature, HSC-like cells and differentiated, monocyte-like cells. The balance of immature and differentiated blasts within a patient is important clinically, as immature AML blasts are acutely sensitive to treatment with venetoclax, a BH3 mimetic that blocks the anti-apoptotic activity of BCL2. BCL2-inhibitor-based therapeutic strategies are the standard of care among elderly patients with untreated or relapsed/refractory AML. While these strategies result in a remission rate of nearly 70%, the majority of patients who do achieve remission ultimately relapse. Frequently, the relapsed disease is composed of differentiated, monocyte-like blasts. Given the importance of BCL2 inhibitors to the clinical management of AML, it is critical to understand the mechanisms by which BCL2 inhibitors result in monocytic relapse. Disease relapse has been traditionally thought to emerge from intrinsically resistant cells that confer a survival advantage under therapeutic pressure. Several studies have suggested that differentiated AML blasts have a survival advantage in the presence of BCL2 inhibition due to a reliance on anti-apoptotic proteins other than BCL2. Immature AML blasts express high levels of BCL2, whereas blasts that are primed for lympho-myeloid or granulocytic-monocytic progenitor cell fates express high levels of MCL-1 and BCL-XL, respectively. However, these studies do not explain why patients with dominant, differentiated subclones frequently achieve long-lasting remission with BCL2-inhibitor-based strategies. Therefore, simple selection of differentiated blasts appears to be an insufficient model to describe BCL2 inhibitor resistance. Alternative models of therapeutic resistance have been identified where cancer cells adapt to a resistant phenotype and acquire a survival advantage under therapeutic pressure. In the context of BCL2 inhibitor resistance, it is unclear the extent to which the emergence of differentiated blasts is the result of selective pressure or blast differentiation state plasticity in response to BCL2 inhibition. In our work, we have investigated the adaptive mechanism of BCL2 inhibition resistance in AML. We have explored this mechanism using an AML cell line model in which immature AML cells continuously generate differentiated AML cells (OCI-AML8227). We used fluorescence-activated cell sorting to isolate immature CD34+/CD38- OCI-AML8227 cells. After 72 hours in liquid culture, we observed that these cells recapitulated a pool of differentiated CD34-/CD38+/CD14+ cells. Furthermore, we used single cell ATAC sequencing to confirm that the unfractionated pool of OCI-AML8227 cells display chromatin accessibility profiles resembling cells across the continuum of myeloid differentiation. To understand whether this model is appropriate to investigate the differentiation-state-specific differences in sensitivity to BLC2 inhibition, we measured the viability of immature CD34+ and differentiated CD34- OCI-AML8227 cells following BCL2 inhibition. Consistent with previous studies, immature CD34+ cells were significantly more sensitive to BCL2 inhibition than differentiated CD34- cells. In addition, BLC2 inhibition of unfractionated OCI-AML8227 cells resulted in an 82% increase in the number of differentiated CD34-/CD38+ cells. While the number of immature CD34+/CD38- cells was reduced following BCL2 inhibition, it was only reduced by 27%. Furthermore, there was no change in the apoptosis marker Annexin V among immature CD34+/CD38- cells. These results support an adaptive resistance model where AML cells acquire resistance through differentiation state plasticity. We will explore this mechanism by following the temporal dynamics of immature and differentiated blasts following BCL2 inhibition. Using single cell barcoding, we will not only be able to trace the trajectories of these populations but also investigate transcriptional adaptation of the immature and differentiated populations. With an understanding of the adaptive mechanism of resistance to BCL2 inhibition, we can nominate alternative targets to prevent the outgrowth of resistance populations and design more effective, rational BLC2-inhibitor-based therapeutic strategies.

Enriched Signalling Pathways in Venetoclax-Relapsed Chronic Lymphocytic Leukemia (CLL) Cells and Targeting Using a Protac-Based Bcl-2/Bcl-Xl Degrader

Venetoclax is a specific inhibitor of Bcl-2, the key protein which protects CLL cells from intrinsic apoptosis, whereas the Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib kills CLL cells via blockade of B-cell receptor (BCR) signalling. Unfortunately, some patients develop treatment resistance after receiving these agents in combination or sequentially. We aimed to evaluate whether CLL cells collected upon venetoclax and ibrutinib relapse retain survival dependency on alternative targets of the BCR pathway and Bcl-2 family, and to propose new vulnerabilities based on gene expression changes. Using single cell RNA sequencing (sc-RNA-seq), we analyzed paired samples from a CLL patient collected at baseline and at relapse, which occurred after cessation of fixed-duration treatment with venetoclax and ibrutinib. The proportion of cells in each CLL cluster was vastly altered at relapse. Cluster 3 represented 4% of CLL cells at baseline versus 36% at relapse and had a high BCR score, including high expression of MAP3K8 FOSB8 and NFKB1 genes. Moreover, at relapse, it contained more CLL cells expressing transcripts from the Bcl-2 family of anti-apoptotic proteins, including Bcl-2 and Bcl-xL. We also integrated sc-RNA-seq data from three primary CLL samples collected at relapse after venetoclax and ibrutinib treatment. The four clusters of CLL cells identified were enriched for BCR, interleukin-mediated, ErbB and the Fc epsilon receptor I signalling pathways. Importantly, most of the relapsed CLL cells which expressed high levels of Mcl-1 or Bcl-xL also expressed Bcl-2, suggesting that these resistant CLL cells became dependent on various anti-apoptotic proteins for survival. These observations led us to test Bcl-2/Bcl-xL targeting PROTACs PZ18753b and WH25244 in venetoclax-resistant CLL. These PROTACs were derived from navitoclax (Bcl-2/Bcl-xL dual inhibitor) linked to a VHL E3 ligase ligand to target Bcl-2 and Bcl-xL for degradation, with improved specificity to cancer cells while sparing platelets. We used three models of venetoclax-resistant CLL: (1) OSU-CLL cells expressing mutant Bcl-2 (G101V, F104L, R107-110dup or A113G), (2) treatment-naïve primary CLL cells co-cultured with NK.Tert stromal cells for 24h (CLL+stroma), and (3) primary CLL cells collected at venetoclax relapse. CLL cells were treated with venetoclax, navitoclax, PZ18753b, WH25244, or negative controls (NC) lacking VHL recruitment, PZ18753b-NC or WH25244-NC. Protein degradation was evaluated by Western Blot; viability, by Cell Titer Glo; and apoptosis, by flow cytometric detection of active BAX and BAK, mitochondrial cytochrome C, and AnnexinV binding. WH25244 was superior to venetoclax at targeting OSU-CLL cells expressing either wildtype or mutant Bcl-2, upon 72h treatment. Cell death by WH25244 was preceded by degradation of Bcl-xL and partial degradation of Bcl-2, despite presence of Bcl-2 mutations. In treatment-naïve primary CLL cells, Bcl-xL degradation by PZ18753b and WH25244 proceeded in a dose-, time- and VHL-dependent manner. The potency of these PROTACs fell in between that of venetoclax and navitoclax, with mitochondrial apoptosis occurring at low nanomolar concentrations known to spare human platelets. Stepwise, activation of BAK and BAX, and cytochrome C release were evident at 14h of treatment, followed by phosphatidyl serine exposure at 18h of treatment. A rightward shift in the dose-response curves of PZ18753b (16-fold) and WH25244 (8-fold) was noted for CLL+stroma cells relative to paired CLL cells treated in suspension. This reduction in potency was associated with (1) uptake of the PROTACs by NK.Tert cells, since Bcl-xL degradation was noted in stromal cells yet without evident toxicity, and (2) up to 2.5-fold increased levels of Bcl-xL in CLL+stroma, which shifted the dose-dependent degradation of Bcl-xL. Finally, 2 out of 3 venetoclax-relapsed primary CLL samples showed more active BAK and cytochrome C release upon treatment with WH25244 than venetoclax, evaluated at 10 nM. In conclusion, WH25244 is a PROTAC-based Bcl-2/Bcl-xL degrader with the potential to overcome venetoclax-resistant CLL dependent on Bcl-xL and mutant Bcl-2. Relative to its precursor, navitoclax, it shows increased potency against CLL cells and decreased toxicity against platelets in vitro, due to its VHL-dependent activity and minimal expression of VHL in platelets.

Synergism of BCL-2 family inhibitors facilitates selective elimination of senescent cells

Accumulation of senescent cells in tissues with advancing age participates in the pathogenesis of several human age-associated diseases. Specific senescent secretome, the resistance of senescent cells to apoptotic stimuli, and lack of immune system response contribute to the accumulation of senescent cells and their adverse effects in tissues. Inhibition of antiapoptotic machinery, augmented in senescent cells, by BCL-2 protein family inhibitors represents a promising approach to eliminate senescent cells from tissues. This study aimed to explore synergistic and selective senolytic effects of anti-apoptotic BCL-2 family targeting compounds, particularly BH3 mimetics.Using human non-transformed cells RPE-1, BJ, and MRC-5 brought to ionizing radiation-, oncogene-, drug-induced and replicative senescence, we found synergy in combining MCL-1 selective inhibitors with other BH3 mimetics. In an attempt to uncover the mechanism of such synergy, we revealed that the surviving subpopulation of cells resistant to individually applied ABT-737/ABT-263, MIK665, ABT-199, and S63845 BCL-2 family inhibitors showed elevated MCL-1 compared to untreated control cells indicating the presence of a subset of cells expressing high MCL-1 levels and, therefore, resistant to BCL-2 inhibitors within the original population of senescent cells.Overall, we found that combining BCL-2 inhibitors can be beneficial for eliminating senescent cells, thereby enabling use of lower, potentially less toxic, doses of drugs compared to monotherapy, thereby overcoming the resistance of the subpopulation of senescent cells to monotherapy.

P317: TARGETING HYPERACTIVE PDGFR-B IN T-ALL AND T-LBL

Background: T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that accounts for 10-15% of pediatric and 25% of adult ALL cases. General prognosis of T-ALL has been improving over time. However, the outcome of T-ALL patients with primary resistant or relapsed leukemia remains dismal. To this end, we have screened a T-ALL/T-LBL patient cohort for copy number variations and identified a novel MYH9-PDGFR-β fusion in a T-LBL case, driving aberrant activation of platelet derived growth factor β (PDGFR-β). Moreover, RNA sequencing data of an independent T-ALL patient cohort showed overexpression of PDGFR-β in the TLX subgroup and in immature T-ALL patients. PDGFR-β is a transmembrane glycoprotein dimer molecule that functions as a receptor tyrosine kinase. Ligand binding results in receptor dimerization and subsequent activation via autophosphorylation, causing downstream activation of various signaling pathways such as PI3K/AKT, MAPK/ERK, JAK/STAT and Notch. As constitutively active PDGFR-β is observed in a variety of malignancies, multi-target kinase inhibitors are already used in the clinic. However, they are not specific and often result in adverse side-effects. CP-673451 is a novel and selective PDGFR inhibitor. Mechanistically, CP-673451 inhibits autophosphorylation of dimeric PDGFR-β and in this way prevents phosphorylation of downstream PI3K/AKT and GSK-3α/β. Previous studies showed growth inhibition of multiple tumor xenografts (lung and colon carcinomas) mainly due to a direct antitumor effect. Aims: We aim to assess whether specific PDGFR-β inhibitors can be used as a novel and targeted therapy in T-ALL/T-LBL treatment or if they can be of added value to the current chemotherapy regimen for T-ALL/T-LBL patients. In the future, we would like to investigate the transforming capacity of the MYH9-PDFR-β fusion in an in vitro pro-T cell culture system. Methods: T-ALL cell lines were screened for PDGFR-β levels and cell lines were selected with both high (CTV-1, SEM) and low (MOLT-16, Jurkat, Loucy) PDGFR-β levels. CP-673451 was tested on the selected cell lines. Cell viability was measured using an ATP assay (CellTiter Glo) and downstream signaling pathways were quantified with flow cytometry and western blot. Additionally, spleen cells from PDGFR-βhigh and PDGFR-βlow T-ALL PDX models were treated ex vivo using CP-673451. Results: Both PDGFR-βhigh cell lines and PDGFR-βhigh PDX models showed sensitivity to CP-673451 treatment in nanomolar range, whereas PDGFR-βlow models did not (Fig. 1A-B). Further characterization of CP-673451 treatment showed inhibition of PDGFR-β autophosphorylation both on western blot and flow cytometry. Downstream, phosphorylation levels of STAT3, STAT5 and GSK-3β were decreased upon PDGFR-β inhibition (Fig. 1C). Dephosphorylation of GSK-3β results in activation of this kinase, leading to phosphorylation of MCL1, promoting degradation of this antiapoptotic protein. Interestingly, high MCL1 levels are associated with lowered sensitivity to glucocorticoids, core components of T-ALL/T-LBL treatment. To this end, combination therapy of PDGFR-β inhibition with glucocorticoids can result in synergistic effects. Next, we will conduct in vivo studies to verify if specific PDGFR-β inhibitors could be advantageous in the treatment of PDGFR-βhigh T-ALL/T-LBL cases. Image:Summary/Conclusion: In this study, we have identified MYH9-PDGFRB as a novel rare fusion activating PDGFR-β in a T-LBL patient sample. Moreover, in vitro evaluation in T-ALL cell lines and ex vivo treatment of T-ALL PDX models showed therapeutic benefits from PDGFR-β inhibition by CP-673451.

Mcl-1 levels critically impact the sensitivities of human colorectal cancer cells to APG-1252-M1, a novel Bcl-2/Bcl-XL dual inhibitor that induces Bax-dependent apoptosis

New treatment options, such as targeted therapies, are urgently needed for the treatment of colorectal cancer (CRC), the third leading cause of cancer-related deaths worldwide. The current study focuses on demonstrating the therapeutic efficacies of APG-1252-M1 (an active form of the prodrug, APG-1252 or pelcitoclax), a highly potent Bcl-2/Bcl-XL dual inhibitor in clinical trials, against CRC and understanding the underlying mechanisms. APG-1252-M1 effectively decreased the survival of CRC cell lines, particularly those expressing relatively low levels of Mcl-1, with the induction of apoptosis. High levels of Mcl-1 were significantly correlated with decreased sensitivity of CRC cell lines to APG-1252-M1. When combined with an Mcl-1 inhibitor, APG-1252-M1 synergistically decreased the survival and induced apoptosis of APG-1252-M1-insensitive cell lines with high levels of Mcl-1. This combination further decreased the survival and enhanced apoptosis even in sensitive cell lines with relatively low levels of Mcl-1, whereas enforced expression of ectopic Mcl-1 in these cells abrogated APG-1252-M1’s effects on decreasing cell survival and inducing apoptosis, which could be reversed by Mcl-1 inhibition. APG-1252-M1 rapidly induced cytochrome C and Smac release from mitochondria with caspase-3 and PARP cleavage. Deficiency of Bax in CRC cells abolished APG-1252-M1’s ability to induce apoptosis, indicating that APG-1252-M1 induces Bax-dependent apoptosis. The current study thus demonstrates the potential of APG-1252-M1 as a monotherapy in the treatment of CRC, particularly those with low Mcl-1 expression, or in combination with an Mcl-1 inhibitor, warranting further evaluation in vivo and in the clinic.

Notch2 Increases the Resistance to Venetoclax-Induced Apoptosis in Chronic Lymphocytic Leukemia B Cells by Inducing Mcl-1.

Chronic lymphocytic leukemia (CLL) has experienced a clinical revolution—thanks to the discovery of crucial pathogenic mechanisms. CLL is still an incurable disease due to intrinsic or acquired resistance of the leukemic clone. Venetoclax is a Bcl-2 inhibitor with a marked activity in CLL, but emerging patterns of resistance are being described. We hypothesize that intrinsic features of CLL cells may contribute to drive mechanisms of resistance to venetoclax. We analyzed the expression of Interferon Regulatory Factor 4 (IRF4), Notch2, and Mcl-1 in a cohort of CLL patients. We evaluated CLL cell viability after genetic and pharmaceutical modulation of Notch2 expression in patients harboring trisomy 12. We tested venetoclax in trisomy 12 CLL cells either silenced or not for Notch2 expression or in combination with an inhibitor of Mcl-1, AMG-176. Trisomy 12 CLL cells were characterized by low expression of IRF4 associated with high levels of Notch2 and Mcl-1. Notch2 and Mcl-1 expression determined protection of CLL cells from spontaneous and drug-induced apoptosis. Considering the involvement of Mcl-1 in venetoclax resistance, our data demonstrated a contribution of high levels of Notch2 and Mcl-1 in a reduced response to venetoclax in CLL cells carrying trisomy 12. Furthermore, reduction of Mcl-1 expression by silencing Notch2 or by treatment with AMG-176 was able to restore the response of CLL cells to venetoclax. The expression of Notch2 identifies a subset of CLL patients, mainly harboring trisomy 12, characterized by high levels of Mcl-1. This biological mechanism may compromise an effective response to venetoclax.

Myeloid cell leukemia-1: a formidable barrier to anticancer therapeutics and the quest of targeting it.

The antiapoptotic B cell lymphoma-2 (Bcl-2) family members are apical regulators of the intrinsic pathway of apoptosis that orchestrate mitochondrial outer membrane permeabilization (MOMP) through interactions with their proapoptotic counterparts. Overexpression of antiapoptotic Bcl-2 family proteins has been linked to therapy resistance and poor prognosis in diverse cancers. Among the antiapoptotic Bcl-2 family members, predominant overexpression of the prosurvival myeloid cell leukemia-1 (Mcl-1) has been reported in a myriad of hematological malignancies and solid tumors, contributing to therapy resistance and poor outcomes, thus making it a potential druggable target. The unique structure of Mcl-1 and its complex regulatory mechanism makes it an adaptive prosurvival switch that ensures tumor cell survival despite therapeutic intervention. This review focusses on diverse mechanisms adopted by tumor cells to maintain sustained elevated levels of Mcl-1 and how high Mcl-1 levels contribute to resistance in conventional as well as targeted therapies. Moreover, recent developments in the Mcl-1-targeted therapeutics and the underlying challenges and considerations in designing novel Mcl-1 inhibitors are also discussed.

Quizartinib (Quiz) with Decitabine (DAC) and Venetoclax (VEN) Is Highly Active in Patients (pts) with FLT3-ITD Mutated Acute Myeloid Leukemia (AML) - RAS/MAPK Mutations Continue to Drive Primary and Secondary Resistance

Quizartinib (Quiz) with Decitabine (DAC) and Venetoclax (VEN) Is Highly Active in Patients (pts) with FLT3-ITD Mutated Acute Myeloid Leukemia (AML) - RAS/MAPK Mutations Continue to Drive Primary and Secondary Resistance

Abstract 977: Mcl-1 levels critically determines the sensitivities of human colon cancer cells to APG1244, a novel Bcl-2 and Bcl-XLinhibitor, that induces Bax- and caspase-8- dependent apoptosis

Abstract Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide due to its poor prognosis. Most of the patients receive surgical resection when diagnosed as CRC, whereas the recurrence rate is very high. Therefore, additional treatment options, such as targeted therapy, are still urgently needed. The novel BH3 mimetic, APG1244 (a prodrug for APG1252), is a highly potent Bcl-2 and Bcl-XL dual inhibitor and being tested in clinical trials as a potential cancer therapeutic agent. The current study focuses on demonstrating the therapeutic efficacies of APG1244 against colorectal cancer and understanding the underlying mechanisms. A panel of CRC cell lines with different levels of Bcl-2, Bcl-XL and Mcl-1 possessed different sensitivities to APG1244. APG1244 effectively decreased the survival of CRC cell lines, particularly those expressing relative low levels of Mcl-1, with induction of apoptosis. High levels of Mcl-1 in these cell lines were significantly correlated to decreased sensitivity to APG1244. APG1244, when combined with a Mcl-1 inhibitor, synergistically decreased the survival and induced apoptosis of APG1244-insensitive cell lines with high levels of Mcl-1. This combination could further decreased the survival and enhanced apoptosis even in the cell lines with relatively low levels of Mcl-1, whereas enforced expression of ectopic Mcl-1 in these cells abrogated APG1244's effects on decreasing cell survival and inducing apoptosis, which can be reversed by Mcl-1 inhibition. In our study, it appears that APG1244 quickly activated caspase-8 and caspase-3 and induced PARP cleavage with induction of cytochrome C and Smac release from mitochondria. Deficiency of Bax, Smac, Bim or PUMA, but not FADD, in CRC cells protected (BAX-KO) or partially protected cells from APG1244-induced apoptosis, indicating that APG1244 induces Bax-dependent apoptosis. Furthermore, blockage of caspase-8 with a caspase-8 specific inhibitor abolished cleavage of caspase-3 and PARP including attenuation of cytochrome C and Smac release induced by APG1244, suggesting a caspase-8-dependent mechanism. The current study thus demonstrates the potential of APG1244 as a monotherapy in the treatment of CRC, particularly those with low Mcl-1 expression, or in combination with a Mcl-1 inhibitor for CRC treatment, warranting further evaluation in vivo and in the clinic. Citation Format: Weilong Yao, Shi-Yong Sun. Mcl-1 levels critically determines the sensitivities of human colon cancer cells to APG1244, a novel Bcl-2 and Bcl-XLinhibitor, that induces Bax- and caspase-8- dependent apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 977.

Abstract 984: Targeting BCL-xL addiction with APG-1252 (pelcitoclax) to overcome apoptotic blockade in neuroendocrine neoplasm (NEN)

Abstract Current targeted therapies such as VEGFR inhibitor sunitinib and mTOR inhibitor everolimus extend progression-free survival (PFS) in patients with Grade 1 (G1) and G2 neuroendocrine tumors (NET), but the objective response rate remains low. The median PFS in patients with advanced neuroendocrine carcinoma (NEC) receiving platinum-based therapy is only 3 to 4 months. Hence, more effective therapy is needed to improve clinical outcomes. This study explored if BCL-2 family antideath proteins play a role in NEN tumorigenesis and whether clinical-stage dual BCL-2/BCL-xL inhibitor APG-1252 might overcome intrinsic apoptotic blockade in NEN. A panel of six NEN cell lines were exposed to APG-1252-M1, which is an active metabolite of APG-1252 typically used for in vitro studies. After 72 hours, the IC50 of APG-1252-M1 ranged from 0.43 to 10 μM. Among the panel, pancreatic NET (pNET) cell lines BON-1 and β-TC3 exhibited the highest sensitivity, with IC50 values of 0.43 and 0.55 µM, respectively, whereas NEC cell line NCI-H460 was insensitive to APG-1252-M1. Interestingly, Western blot analysis revealed higher expression of BCL-xL compared to BCL-2 and MCL-1 proteins in NET cell lines (BON-1, QGP-1, NCI-H727). Higher baseline levels of BCL-xL:BIM and BCL-xL:PUMA complexes in NEN cells indicated APG-1252-M1 sensitivity. Exposure to APG-1252-M1 disrupted these complexes, liberating BIM/PUMA to trigger the apoptotic cascade, as indicated by an increase in BAX:BAK complexes. Conversely, lower BCL-xL complex levels and higher MCL-1 levels correlated with insensitivity of NCI-H460 to APG-1252-M1. Immunohistochemistry staining of BCL-xL, BCL-2, and MCL-1 proteins was performed in formalin-fixed paraffin-embedded tissue sections of 106 NEN patient tumors (including stomach/intestine/pancreatic NEN), of which approximately 70% were NET and 30% NEC. Similar to data in NEN cell lines, BCL-xL expression was higher than BCL-2 expression in NEN patient samples, in which BCL-xL expression was indicated by more than 80% of samples with IHC scoring ≥6 on a scale of 0 to 10, and BCL-2 expression by 30% IHC scoring < 6 or undetectable (60% IHC scoring 0). Unexpectedly, however, MCL-1 expression was also high, with more than 80% of samples IHC scoring ≥6. In summary, the results suggest that BCL-xL plays an important role in NEN. Cellular sensitivity to BCL-2/BCL-xL inhibitor APG-1252-M1 correlates with baseline BCL-xL complex levels. In NEN patient samples, MCL-1 was also highly expressed, implicating its potential negative regulatory effect on sensitivity to APG-1252. Concurrent expression of BCL-xL and MCL-1 proteins suggests that a combination treatment targeting both proteins might be more effective in NEN. Our findings inform development of a BCL-2/BCL-xL inhibitor for NEN therapy. Citation Format: Yu Guo, Jing Deng, Lin Zhang, Xiaojing Huang, Kaixiang Zhang, Feng Zhou, Yuanbao Li, Fan Luo, Douglas D. Fang, Dajun Yang, Jie Chen, Yifan Zhai. Targeting BCL-xL addiction with APG-1252 (pelcitoclax) to overcome apoptotic blockade in neuroendocrine neoplasm (NEN) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 984.

High-Dimensional Single-Cell Proteomic Analysis Reveals Therapeutic Vulnerabilities in Relapsed/Refractory AML By Concomitant Analysis of Cell Surface Antigens, Signaling Pathways and Anti-Apoptotic Proteins

Background: The heterogeneous and adaptive nature of AML-associated genomic and proteomic landscape may account for disease relapse and poor prognosis, as therapy-associated selective pressure drives the emergence and expansion of AML clones with features different from those detected at diagnosis. The evolving focus is on single-cell analytical tools to fully capture the pathobiological heterogeneity of AML. We leveraged CyTOF to interrogate the proteomic heterogeneity in patients with R/R AML, which could potentially permit the design of rational combinatorial therapeutic approaches targeting vulnerabilities in these cells.. Methods: To dissect AML heterogeneity and its contribution to treatment failure, we designed a 51-parameter CyTOF panel and interrogated cellular hierarchy, major immune phenotypes, anti-apoptotic molecules, signaling pathways, exhaustion markers and attractive targets for CAR T-cell therapy in R/R AML (n:13). Results: First, we generated two-dimensional t-SNE maps and observed that the leukemic bone marrow compartment harbored immature (CD34+CD38- and CD34+CD38+) and mature leukemic blasts (CD33+CD34-) and major immune subsets. Constitutively active signaling pathways characterized by high levels of p-4EBP1, p-MEK1/2, p-S6 and p-AKT, marked immature and mature leukemia cells and comparative analysis revealed that monocytic blasts harbored more active signaling networks. The proportions of these subpopulations varied significantly across patients. We initially assessed the distribution of anti-apoptotic molecules across these leukemia compartments. Strikingly, Bcl-2 levels were considerably high within less-differentiated leukemic cell compartments and CD68 expressing leukemic blasts with monocytic differentiation had significantly lower levels of Bcl-2. This suggests that differentiated leukemic cells could preferentially survive under selection pressure of Bcl-2 inhibitors. On the contrary, we observed a trend towards higher Mcl-1 levels in differentiated leukemia cells. These findings provide a rationale for combining therapeutic modalities to target different leukemia subpopulations. Indeed, Bcl-2 and Mcl-1 inhibitors (Venetoclax and AZD5991) resulted in highly synergistic effects in AML PDX models. Hence, this analysis supports the hypothesis that Mcl-1 overexpression is a resistance factor to Bcl-2 inhibition, usually understood as developing in the same cell. Next, we assessed expression patterns of putative CAR T-cell targets expressed on leukemic cells and identified significant variegated expression patterns in R/R AML samples. CD123 demonstrated patchy distribution across immature and differentiated leukemic blasts while CD33 expression was the main characteristic of differentiated leukemic blasts. Of note, the immature leukemia compartment demonstrated variable levels of CD33 and complete lack of CD33 on CD34+ leukemic cells was observed in a subgroup of patients. CLL-1 was uniformly expressed across all leukemia compartments, but was not ubiquitously expressed in all patient samples, revealing substantial interpatient heterogeneity in R/R AML and highlighting the concept of targeting at least two antigens concomitantly. Lastly, we sought to undertake high-dimensional assessment of immune compartments to identify major immune phenotypes in heavily treated R/R AML patients and discover the link between immune phenotypes and AML-associated traits. In line with leukemia cell heterogeneity, we found a significant degree of variation in immune cell composition among patients. Inhibitory molecules PD-1 and TIGIT were significantly expressed on CD4 and CD8 T-cells respectively, providing a rationale for use of combinatorial immunotherapeutic approach for the treatment of AML. Conclusion: Single-cell profiling of R/R AML using CyTOF reveals significantly heterogeneous expression patterns of molecules targeted by BH3 mimetics (Bcl-2 and Mcl-1), CAR T-cells, and other antibody-based immunotherapeutic therapies. This approach provides a rationale to develop combinatorial therapeutic approaches targeting distinct leukemia sub-populations with discrete expression patterns of established and novel putative targets. An example is the combined targeting of Bcl-2 and Mcl-1, which are differentially expressed in early and more differentiated leukemia subpopulations. Disclosures Carter: AstraZeneca: Research Funding; Syndax: Research Funding; Amgen: Research Funding; Ascentage: Research Funding. Andreeff:Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Amgen: Research Funding.

Targeting Mcl-1 Enhances the Activity of Tyrosine Kinase Inhibitor Gilteritinib in FLT3 Mutated AML

Anti-apoptotic Bcl-2 proteins play critical roles in AML cell and AML stem/progenitor cell survival and drug resistance, hence are relevant therapeutic targets. Indeed, the combination of the selective Bcl-2 inhibitor venetoclax (VEN) with a hypomethylating agent elicits CR/CRi rates of > 65%, is well tolerated by elderly AML patients, and obtained FDA approval. However, despite of the major improvement in response rates, survival extension was limited and most patients ultimately relapsed largely due to the development of resistant disease. Molecular analysis of treated patients revealed that primary and adaptive resistance to VEN-based combinations was frequently characterized by acquisition or enrichment of clones activating signaling pathways such as FLT3 or RAS (DiNardo CD et al., Blood 2020). FLT3 is one of the most frequently mutated gene in AML, resulting in constitutive activation of FLT3 tyrosine kinase and its downstream signaling pathways such as RAS/MAPK, which can be targeted by FLT3 tyrosine kinase inhibitors (TKIs). However, patients treated with TKIs ultimately relapse and adapt to TKI therapy by reactivating the MAPK signaling pathway (Bruner JK et al., Cancer Res 2017), which is known to stabilize Mcl-1 levels. Furthermore, deregulated Mcl-1 expression was identified as a novel mechanism of primary TKI resistance in a subset of FLT3-ITD mutated AML patients (Breitenbuecher F et al., Blood 2009). Importantly, Mcl-1 expression can be induced by VEN treatment and is a major resistance factor to VEN (Pan R et al., Cancer Discover 2014; Carter BZ et al., ASH 2018). Hence, Mcl-1 inhibition may enhance the efficacy of TKIs in FLT3 mutated AML, targeting AML cells and stem/progenitor cells. To determine if targeting Mcl-1 enhances the activity of TKIs in FLT3 mutated AML, we treated MV4-11 and Molm13 cells with Mcl-1 inhibitor AMG176 and TKI gilteritinib (GIL) and observed synergism, as defined by combination index < 1 in both cells. Mechanistic studies demonstrated that GIL markedly decreased Mcl-1 and antagonized AMG176-induced Mcl-1 induction. GIL and its combination with AMG176 also decreased Bcl-xL. Although Bcl-2 protein levels were largely not changed in MV4-11 cells, we found both single treatment and the combination greatly decreased Bcl-2 associated athanogene (BAG) proteins BAG1, BAG3, and BAG4 at the RNA level, which needs to be confirmed at the protein level. The BAG proteins are a family of chaperone regulators and BAG1 was reported to bind and enhance the activity of multiple proteins known to support cells survival, including Bcl-2 (Takayama S et al., Cell 1995). Interestingly, GIL treatment greatly diminished the levels of beta-catenin and its target protein c-Myc, consistent with our previous report that FLT3 regulates beta-catenin signaling (Xiang et al., CCR, 2018). We have generated Mcl-1 overexpressing (OE) and VEN-resistance (VEN-R) MV4-11 and Molm13 cells. The Mcl-1 OE cells are highly resistant to VEN and the VEN-R cells expressed high levels of Mcl-1. Combined inhibition of AMG176 and GIL synergistically induced cell death in Mcl-1 OE and VEN-R resistant cells. Although the expression is low in AML cells we tested, BCL2A1 is also known as a resistant factor to VEN. We generated BCL2A1 OE MV4-11 and Molm13 cells and demonstrated that combined inhibition of FLT3 and Mcl-1 was highly effective in these cells as well. Western blot analysis revealed that GIL effectively decreased Mcl-1 in Mcl-1 OE and VEN-R and BCL2A1 in BCL2A1 OE MV4-11 cells. Next, we treated FLT3 mutated AML patient samples harboring both, ITD and D835 mutations, from 2 patients who had both failed VEN-based therapy and from 1 patient with ITD mutation, with AMG176 and GIL under MSC co-culture conditions. Synergy was observed in all samples in AML blasts and AML stem/progenitor cells. Collectively, our data demonstrate that targeting Mcl-1 enhances the activity of GIL in FLT3 mutated AML, including those resistant to/relapsed from VEN-based therapy, findings that may warrant clinical evaluation. Disclosures Carter: Syndax: Research Funding; Ascentage: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding. Hughes:Amgen: Current Employment. Chen:Amgen: Current Employment. Morrow:Amgen: Current Employment. Andreeff:Amgen: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

AML-354: High-Dimensional Single-Cell Proteomic Analysis Reveals Therapeutic Vulnerabilities in Patients with Relapsed/Refractory AML

We designed a 51-parameter mass cytometry panel and interrogated acute myeloid leukemia (AML) heterogeneity to assess cellular hierarchy, anti-apoptotic molecules, exhaustion markers and targets for CAR T-cell therapy in R/R AML. We generated two-dimensional t-SNE maps to visualize distinct populations and observed significant heterogeneity among patients with regards to leukemic compartment composition. Notably, the leukemic bone marrow compartment harbored leukemia stem cells (CD34+CD38-), immature leukemic cells (CD34+CD38+), more differentiated leukemic blasts (CD33+CD34-) and major immune subsets. We initially assessed the distribution of anti-apoptotic molecules across leukemia compartments. Strikingly, Bcl-2 levels were considerably high within less-differentiated leukemic cell compartments and leukemic blasts with monocytic differentiation had significantly low levels of Bcl-2. On the contrary, we observed a trend towards higher Mcl-1 levels in differentiated leukemic cells. These findings provide a rationale for combining therapeutic modalities to target different leukemia subpopulations. Indeed, Bcl-2 and Mcl-1 inhibitors resulted in highly synergistic effects in AML PDX models (Carter et.al 2018, 2019). Of interest, Bcl-xl was ubiquitously expressed across leukemic and immune cell clusters. Next, we assessed expression patterns of CAR T-cell targets expressed on leukemic cells and identified significant variegated expression patterns for all CAR T-cell therapy targets in R/R AML samples. CD123 demonstrated patchy distribution across immature and differentiated leukemic blasts while CD33 expression was the main characteristic of differentiated leukemic blasts. Of note, the immature leukemia compartment demonstrated variable levels of CD33 and complete lack of CD33 on CD34+ leukemic cells was observed in a subgroup of patients. CLL-1 expression was the most promising marker and expressed across almost all leukemia compartments. Nevertheless, CLL-1 expression on leukemic cells was not ubiquitous in a small group of patients. In line with leukemic cell heterogeneity, we found a significant degree of variation in immune cell composition. Inhibitory molecules PD-1 and TIGIT were significantly expressed on CD4 and CD8 T-cells respectively. Single-cell profiling of R/R AML using mass cytometry reveals significant heterogeneous expression patterns of molecules targeted by CAR T-cells, immunotherapeutic approaches and targeted therapies. This approach provides a rationale to adopt combinatorial therapeutic approaches to target distinct leukemic populations with widely varying target expression.

Abstract 6217: Synergistic effect of CDK8 and BCL-2 inhibition in AML through regulation of MCL-1 and BIM balance

Abstract Background Acute myeloid leukemia (AML) is characterized by rapid proliferation of myeloid blood cells. Due to its heterogeneity and the high rate of acquired drug resistance, new treatment modalities are needed. SEL120 is a specific type I selective inhibitor of Cyclin-dependent kinase 8 (CDK8) and Cyclin-dependent kinase 19 (CDK19). A first- in- human phase Ib clinical trial with SEL120 in patients with AML or HR-MDS was initiated in June 2019. Preclinical studies demonstrated high efficacy of SEL120 in experimental AML models via mechanisms involving differentiation and induction of apoptosis. Transcriptomic analysis of hematological cell lines demonstrated that SEL120 treatment upregulated expression of an apoptotic activator BIM from BCL-2 family of proteins. Methods Efficacy of the compound alone or in combination was tested in viability assays in a broad panel of cancer cell lines. Activity and mechanism of action of CDK8 inhibitor - SEL120 alone and in combination was investigated by flow cytometry, western blotting, co-immunoprecipitation, differential gene expression and ChIPseq analysis. In vivo efficacy was tested in mice injected with MV4-11 cell line both subcutaneously and intravenously. Results Here we provide a strong rationale for combination of SEL120 and BCL-2-selective inhibitor Venetoclax (ABT-199). We found that SEL120 synergistically induced apoptosis with Venetoclax in AML cells. Combination of both compounds significantly reduced levels of prosurvival protein MCL-1 and induced hallmarks of apoptosis including Caspase-3 activation and PARP cleavage. Previous studies associated Venetoclax resistance with increased sequestration of proapoptotic BIM by high levels of MCL-1. While a SEL120 treatment alone had no effects on MCL-1 levels, combination of both compounds resulted in sensitization of Venetoclax-resistant AML cells. We demonstrated that mechanism of Venetoclax resistance can be abrogated by the cotreatment with SEL120 leading to changes in proportions of BIM and MCL-1 levels. Synergistic interaction between SEL120 and Venetoclax resulted in apoptotic cell death in established cell lines and patient derived AML cells. Finally, using murine models of subcutaneous or disseminated AML, we found complete remissions of AML and associated recovery of normal cells in bone marrow of animals treated with both SEL120 and Venetoclax. Conclusion Taken together, these data provided rationale for a novel clinical strategy that may lead to durable responses in AML patients. Citation Format: Tomasz Rzymski, Marta Obacz, Milena Mazan, Marion Chappelier, Marcus Järås, Michal Mikula, Elżbieta Adamczyk, Katarzyna Wiklik, Michal Combik, Aniela Golas, Magdalena Masiejczyk, Przemyslaw Juszczynski, Jerzy Ostrowski, Krzysztof Brzózka. Synergistic effect of CDK8 and BCL-2 inhibition in AML through regulation of MCL-1 and BIM balance [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 6217.

NOTCH2 Contributes to Venetoclax Resistance in Chronic Lymphocytic Leukemia

BACKGROUND: Chronic lymphocytic leukemia (CLL) has recently experienced an unprecedented revolution thanks to the discovery of crucial pathogenetic mechanisms. Despite considerable therapeutic advancements, the eradication of the disease is not complete and resistance and transformation may occur. Venetoclax is a small-molecule BH3 mimetic that competes for binding in the hydrophobic groove of Bcl-2, a key protein involved in CLL cells survival. Venetoclax was shown to have an excellent antitumor activity in patients with relapsed CLL including those with high risk features. However, venetoclax monotherapy shows an overall response rate of 79% and complete response rates of 16% and leave some open questions mainly related to intrinsic features of CLL patients that may guide a pattern of resistance. A recent work shows that durable response to venetoclax based therapy is more likely in patients having minimal adenopathy, mutated IGHV gene, wild type TP53 and Notch1 genes. AIM OF THE WORK: We hypothesize that specific intrinsic features of CLL cells may contribute to drive possible mechanisms of resistance to venetoclax (ABT-199) treatment. METHODS: Notch2 expression was monitored by western blot in purified CLL cells. Modulation of Notch2 expression in vitro was performed by using siRNA strategy. ABT-199 was used at doses of 0.1 nM or 1 nM. RESULTS: Notch signaling is relevant in CLL pathogenesis. We detected a peculiar high expression of Notch2 in a subgroup of CLL patients carrying trisomy 12. The high expression of Notch2 correlated with high levels of Mcl-1, CD23 and Hes1. Interfering with Notch2 expression in trisomy 12 CLL patients, by siRNA silencing, was able to affect leukemic cells viability, reducing CD23 and Mcl-1 expression. Since Mcl-1 is involved in ABT-199 resistance, we wondered if ABT-199 may have a different effect in CLL cells isolated from patients carrying trisomy 12 comparing to no trisomy 12 cases. After 24h of culture in complete medium, trisomy 12 CLL cells showed a gain in survival rate of 10% during treatment with both 0.1 nM or 1 nM in comparison to no trisomy 12 cases. This advantage in viability reflected the maintenance of Notch2 and Mcl-1 expression in trisomy 12 both at 0.1 nM and 1 nM of ABT-199 compared to control. Conversely, a reduction of Notch2 and consequently Mcl-1 levels were observed in no trisomy 12 cases at both doses of ABT-199. We did not detect any variation in Bcl-2 levels. We wondered if Notch2 expression may reduce the response to ABT-199 in trisomy 12 CLL. To demonstrate this hypothesis, we interfered with Notch2 by silencing strategy. Treatment with Notch2 siRNA decreased the expression of Notch2 and Mcl-1 in combination with ABT-199 as shown in Figure 1A. We also found that Notch2 down-regulation cooperated with ABT-199 decreasing CLL cells viability (Figure 1B). CONCLUSIONS: Collectively, our results show a novel mechanism that may compromise the clinical response to venetoclax in CLL patients. Although the excellent mechanism of action of venetoclax, Bcl-XL and Mcl-1, two major anti-apoptotic proteins of Bcl-2 family not inhibited by venetoclax, are key determinants of both acquired and intrinsic resistance to treatment. The possibility to identify patients with more pronounced expression of Mcl-1 may help to optimize the treatment. The expression of Notch2 identify a subset of CLL patients, mainly harboring trisomy 12 aberration, that through the maintenance of high levels of Mcl-1, may be involved in a reduced response to ABT-199. Disclosures Luppi: Gilead Sci., MSD, Pfizer, Novartis, Abbvie, Sanofi, Daiichi Sankyo, Jazz Pharmaceuticals: Honoraria. Marasca:Janssen and Gilead Sci, Abbvie, Roche and Shire: Honoraria, Research Funding.

Abstract P5-03-06: Overcoming MEK inhibitor resistance in triple-negative breast cancer by targeting myeloid cell leukemia-1 (MCL1), an anti-apoptotic protein

Abstract Background: Triple-negative breast cancer (TNBC), which affects over 170 000 women worldwide every year, is considered the most arduous to treat subtype of breast cancer. With no targeted therapy, high rates of drug resistance and rapid metastasis, TNBC carries a poor prognosis. The MEK-ERK-MAPK signaling cascade is known to play a role in numerous cancers. Despite the lack of activating Ras/MAPK mutations in breast cancer, transcriptional signatures of this pathway are prevalent in TNBC. Our previous work showed that TNBC patients with tumors overexpressing ERK2 had a lower overall survival rate than did patients with low-ERK2-expressing tumors. MEK inhibitors selumetinib (AZD6244) and pimasertib (AS703026) are active in preclinical models, but not as single agents in the clinic. Using a synthetic lethal siRNA screen, we identified myeloid cell leukemia-1 (MCL1) as a potential contributor to selumetinib resistance. Mcl-1 is an anti-apoptotic protein that is highly amplified in numerous human cancers. It is associated with cell immortalization, transformation, and chemoresistance. Patients with TNBC tumors expressing high levels of Mcl-1 have lower overall survival and distant-metastasis-free survival rates. We hypothesized that Mcl-1 promotes MEK inhibitor resistance in TNBC. Methods/Results: To model MEK inhibitor resistance, we established selumetinib- and pimasertib-resistant clones of SUM-149 and MDA-MB-231 TNBC cells by continuous exposure to increasing concentrations of inhibitors over a six month period. We confirmed the onset of MEK resistance by demonstrating that resistant cells, in comparison to the parental cells, exhibited no change in cell proliferation upon treatment with the MEK inhibitors. Resistant cells also displayed more effective cell migration and mammosphere formation than parental cells, suggesting a higher fraction of tumor-initiating cells. We found Mcl-1 to be highly expressed in 83% (15 of 18) of TNBC cell lines but only 30% (3 of 10) of other breast cancer cell lines. Resistant cells had higher levels of Mcl-1 than did parental cells. To determine whether Mcl-1 is required for MEK sensitivity, we treated parental and resistant cells with either selumetinib or pimasertib together with S63845, a highly specific Mcl-1 inhibitor. The Mcl-1 inhibitor restored MEK sensitivity in both resistant cell lines. After treatment with the Mcl-1 inhibitor, the resistant SUM-149 and MDA-MB-231 cells had similar cell proliferation rates to those of their parental counterparts. Similar studies were done using an siRNA against Mcl-1. Conclusion: Our data demonstrate that Mcl-1 may promote TNBC resistance to MEK inhibitors and that Mcl-1 is a promising target for combination therapy. We will continue to explore the mechanisms of MEK inhibitor resistance by screening for additional genes/pathways involved. Our long-term goal is to design rational combination approaches to counteract the emergence of resistance by using novel molecularly targeted therapeutics. Citation Format: Gagliardi M, Chauhan G, Pitner MK, Iles L, Qi Y, Pusztai L, Tripathy D, Bartholomeusz G, Bartholomeusz C. Overcoming MEK inhibitor resistance in triple-negative breast cancer by targeting myeloid cell leukemia-1 (MCL1), an anti-apoptotic protein [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-06.

PO-027 Potential use of BH3 mimetics in the treatment of head and neck cancer

IntroductionHead and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. The limited success and significant toxicities associated with current therapies highlight the need for more efficacious treatment strategies. BH3 mimetics are an emerging class of drugs that target the anti-apoptotic BCL-2 family members, which are overexpressed proteins in many cancers, contributing to evasion of apoptosis and resistance to many therapeutic regimens. The BCL-2 specific inhibitor, venetoclax, is currently used to treat haematological malignancies, whereas specific inhibitors for BCL-XL and MCL-1 are entering clinical trials. We aim to determine the potential of BH3 mimetics in the treatment of HNSCC.Material and methodsHNSCC tissue microarrays (TMAs) were immunohistochemically stained for BCL-2, BCL-XL and MCL-1, protein levels assessed semi-quantitatively as low, moderate or high and correlated with patient survival. Cell lines and primary cells were treated with ABT-199 (BCL-2 specific), A1331852 (BCL-XL specific) and/or S63845 (MCL-1 specific), and apoptosis induction and replicative potential were assessed.Results and discussionsTMAs revealed that HNSCC tumours express moderate to high levels of BCL-XL and MCL-1, but low levels of BCL-2. Indeed treatment with a BCL-2 inhibitor had little effect on cell death in HNSCC cell lines; nor did treatment with A1331852 or S63845 as single agents. However, a combination of MCL-1 and BCL-XL inhibition induced extensive apoptotic cell death and a marked reduction in clonogenic cell growth, suggesting a dependence on these two anti-apoptotic proteins for tumour cell survival. A comparison of MCL-1 and BCL-XL expression levels against overall survival of HNSCC patients over a five year period post-surgery revealed that high MCL-1 levels were associated with low median survival. In contrast, the correlation between high BCL-XL expression and median survival was statistically not significant, although it may be clinically relevant.ConclusionBCL-2 is poorly expressed in HNSCC tumours, diminishing the potential of using venetoclax in HNSCC. In contrast, both BCL-XL and MCL-1 are highly expressed in HNSCC, and may be important prognostic indicators. Therefore targeting BCL-XL and MCL-1 together may be therapeutically beneficial, although the toxicity of this combination must be assessed before human trials can be considered.

JDP2: An oncogenic bZIP transcription factor in T cell acute lymphoblastic leukemia.

A substantial subset of patients with T cell acute lymphoblastic leukemia (T-ALL) develops resistance to steroids and succumbs to their disease. JDP2 encodes a bZIP protein that has been implicated as a T-ALL oncogene from insertional mutagenesis studies in mice, but its role in human T-ALL pathogenesis has remained obscure. Here we show that JDP2 is aberrantly expressed in a subset of T-ALL patients and is associated with poor survival. JDP2 is required for T-ALL cell survival, as its depletion by short hairpin RNA knockdown leads to apoptosis. Mechanistically, JDP2 regulates prosurvival signaling through direct transcriptional regulation of MCL1. Furthermore, JDP2 is one of few oncogenes capable of initiating T-ALL in transgenic zebrafish. Notably, thymocytes from rag2:jdp2 transgenic zebrafish express high levels of mcl1 and demonstrate resistance to steroids in vivo. These studies establish JDP2 as a novel oncogene in high-risk T-ALL and implicate overexpression of MCL1 as a mechanism of steroid resistance in JDP2-overexpressing cells.

Aspirin restores ABT-737-mediated apoptosis in human renal carcinoma cells

Aspirin is a novel chemopreventive agent against malignancy. However, outcomes of aspirin monotherapy of renal cell carcinoma (RCC) are inconsistent across studies. ABT-737, an BH3 mimetic inhibitor, is also a promising antitumor drug. Cancer cells including those from RCC, that have high levels of Mcl-1, are refractory to ABT-737-induced apoptosis. We here investigated how aspirin treatment modulates the ABT-737-induced apoptosis. Using the in vitro model of human 786-O cells, we showed that aspirin had sensitized cells to ABT-737 induced apoptosis. Such aspirin-induced changes of ABT-737 resistance was accompanied by a host of biochemical events like protein phosphatase 2A (PP2A) activation, AKT dephosphorylation, Mcl-1/FLICE inhibiting protein (FLIP)/XIAP downregulation, and Bax mitochondrial redistribution. The PP2A inhibitor, okadaic acid, was able to reverse the apirin-induced apoptotic changes. Apart from the aspirin treatment, Mcl-1 silencing also rendered cells vulnerable to ABT-737 induced apoptosis. Since PP2A, Akt, and Mcl-1 play critical roles in RCC malignancy and treatment resistance, our present study showed that aspirin, an alternative adjuvant agent, had recalled ABT-737 sensitivity in the RCC cells through processes involving the PP2A/Akt/Mcl-1 axis.

Abstract 1106: Alvocidib potentiates the activity of venetoclax in preclinical models of multiple myeloma

Abstract The proteasome inhibitor bortezomib is widely used in the treatment of patients with multiple myeloma (MM). The expression levels of many proteins increase as a result of bortezomib treatment, including the pro-apoptotic protein NOXA. NOXA functions to sequester the anti-apoptotic BCL-2 family member, MCL-1. High levels of MCL-1 and/or low levels of NOXA have been implicated in bortezomib resistance and negative patient outcomes, including short duration of treatment response. The BCL-2-specific BH3 mimetic venetoclax (ABT-199) has also been explored in multiple hematological malignancies, including the treatment of MM. Venetoclax induces apoptosis in a BCL-2 specific manner by directly inhibiting BCL-2 function. However, intrinsic resistance to venetoclax treatment observed in MM patient samples has been attributed to a low BCL-2-to-MCL-1 gene expression ratio, suggesting a central role for MCL-1 in cell survival in this context as well. Increased MCL-1 expression is a known resistance mechanism to venetoclax treatment in a variety of cell types including chronic lymphocytic leukemia and lymphomas. Considering the central role of MCL-1 to treatment efficacy in MM, we investigated the ability of an MCL-1-lowering agent, namely the CDK9 inhibitor alvocidib, to potentiate the activity of venetoclax in MM. Alvocidib suppresses MCL-1 expression via CDK9-mediated regulation of RNA polymerase II. Alvocidib has achieved robust improvements in the clinical response rates of high-risk, newly diagnosed acute myeloid leukemia (AML) patients as part of the time-sequential ACM regimen (alvocidib + cytarabine + mitoxantrone). We therefore hypothesized that alvocidib would potentiate the activity of venetoclax in MM through an MCL-1-dependent mechanism. In this report, we demonstrate that alvocidib inhibits the protein expression of MCL-1 in MM cells in a time-dependent fashion, up to 96 hours. In cell viability assays, the addition of up to 100 nM venetoclax resulted in a 2.8-fold reduction in the IC50 of alvocidib in the cultured OPM-2 cell line. Conversely, the potentiation of venetoclax activity with the addition of alvocidib resulted in a more than 500-fold decrease in IC50 in the relatively venetoclax-resistant OPM-2 cells. Additional studies are currently underway to investigate the efficacy of alvocidib and venetoclax in the context of bortezomib resistance where low NOXA may contribute to enhanced cell survival via MCL-1. Taken together, our data suggest that the combination of alvocidib with venetoclax may constitute a novel therapeutic regimen in the treatment of MM. Further, it suggests that CDK9-mediated targeting of MCL-1 may offer a clinical route to addressing intrinsic resistance in MM patients. Citation Format: Mark Livingston, Wontak Kim, Hillary Haws, Peter Peterson, Clifford J. Whatcott, Adam Siddiqui-Jain, Steven Weitman, David J. Bearss, Steven L. Warner. Alvocidib potentiates the activity of venetoclax in preclinical models of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1106. doi:10.1158/1538-7445.AM2017-1106