Metabolic reprogramming represents a targetable mechanism to overcome acquired resistance to venetoclax in acute myeloid leukemia.

Treatment Outcomes with Hypomethylating Agents and Venetoclax for AML in the Community Compared to an Academic Setting

Focal adhesion kinase (FAK) promotes cancer cell growth and metastasis. We previously reported that FAK inhibition by the selective inhibitor VS-4718 exerted antileukemia activities in acute myeloid leukemia (AML). The mechanisms involved, and whether VS-4718 potentiates efficacy of other therapeutic agents, have not been investigated. Resistance to apoptosis inducted by the BCL-2 inhibitor ABT-199 (venetoclax) in AML is mediated by preexisting and ABT-199-induced overexpression of MCL-1 and BCL-XL. We observed that VS-4718 or silencing FAK with siRNA decreased MCL-1 and BCL-XL levels. Importantly, VS-4718 antagonized ABT-199-induced MCL-1 and BCL-XL. VS-4718 markedly synergized with ABT-199 to induce apoptosis in AML cells, including primary AML CD34+ cells and AML cells overexpressing MCL-1 or BCL-XL. In a patient-derived xenograft (PDX) model derived from a patient sample with NPM1/FLT3-ITD/TET2/DNMT3A/WT1 mutations and complex karyotype, VS-4718 statistically significantly reduced leukemia tissue infiltration and extended survival (72 vs. control 36 days, P = 0.0002), and only its combination with ABT-199 effectively decreased systemic leukemia tissue infiltration and circulating blasts, and prolonged survival (65.5 vs. control 36 days, P = 0.0119). Furthermore, the combination decreased NFκB signaling and induced the expression of IFN genes in vivo The combination also markedly extended survival of a second PDX model developed from an aggressive, TP53-mutated complex karyotype AML sample. The data suggest that the combined inhibition of FAK and BCL-2 enhances antileukemia activity in AML at least in part by suppressing MCL-1 and BCL-XL and that this combination may be effective in AML with TP53 and other mutations, and thus benefit patients with high-risk AML.

<div>Abstract<p>Focal adhesion kinase (FAK) promotes cancer cell growth and metastasis. We previously reported that FAK inhibition by the selective inhibitor VS-4718 exerted antileukemia activities in acute myeloid leukemia (AML). The mechanisms involved, and whether VS-4718 potentiates efficacy of other therapeutic agents, have not been investigated. Resistance to apoptosis inducted by the BCL-2 inhibitor ABT-199 (venetoclax) in AML is mediated by preexisting and ABT-199–induced overexpression of MCL-1 and BCL-XL. We observed that VS-4718 or silencing FAK with siRNA decreased MCL-1 and BCL-XL levels. Importantly, VS-4718 antagonized ABT-199–induced MCL-1 and BCL-XL. VS-4718 markedly synergized with ABT-199 to induce apoptosis in AML cells, including primary AML CD34<sup>+</sup> cells and AML cells overexpressing MCL-1 or BCL-XL. In a patient-derived xenograft (PDX) model derived from a patient sample with <i>NPM1/FLT3-ITD/TET2/DNMT3A/WT1</i> mutations and complex karyotype, VS-4718 statistically significantly reduced leukemia tissue infiltration and extended survival (72 vs. control 36 days, <i>P</i> = 0.0002), and only its combination with ABT-199 effectively decreased systemic leukemia tissue infiltration and circulating blasts, and prolonged survival (65.5 vs. control 36 days, <i>P</i> = 0.0119). Furthermore, the combination decreased NFκB signaling and induced the expression of IFN genes <i>in vivo</i>. The combination also markedly extended survival of a second PDX model developed from an aggressive, <i>TP53</i>-mutated complex karyotype AML sample. The data suggest that the combined inhibition of FAK and BCL-2 enhances antileukemia activity in AML at least in part by suppressing MCL-1 and BCL-XL and that this combination may be effective in AML with <i>TP53</i> and other mutations, and thus benefit patients with high-risk AML.</p></div>

<div>Abstract<p>Focal adhesion kinase (FAK) promotes cancer cell growth and metastasis. We previously reported that FAK inhibition by the selective inhibitor VS-4718 exerted antileukemia activities in acute myeloid leukemia (AML). The mechanisms involved, and whether VS-4718 potentiates efficacy of other therapeutic agents, have not been investigated. Resistance to apoptosis inducted by the BCL-2 inhibitor ABT-199 (venetoclax) in AML is mediated by preexisting and ABT-199–induced overexpression of MCL-1 and BCL-XL. We observed that VS-4718 or silencing FAK with siRNA decreased MCL-1 and BCL-XL levels. Importantly, VS-4718 antagonized ABT-199–induced MCL-1 and BCL-XL. VS-4718 markedly synergized with ABT-199 to induce apoptosis in AML cells, including primary AML CD34<sup>+</sup> cells and AML cells overexpressing MCL-1 or BCL-XL. In a patient-derived xenograft (PDX) model derived from a patient sample with <i>NPM1/FLT3-ITD/TET2/DNMT3A/WT1</i> mutations and complex karyotype, VS-4718 statistically significantly reduced leukemia tissue infiltration and extended survival (72 vs. control 36 days, <i>P</i> = 0.0002), and only its combination with ABT-199 effectively decreased systemic leukemia tissue infiltration and circulating blasts, and prolonged survival (65.5 vs. control 36 days, <i>P</i> = 0.0119). Furthermore, the combination decreased NFκB signaling and induced the expression of IFN genes <i>in vivo</i>. The combination also markedly extended survival of a second PDX model developed from an aggressive, <i>TP53</i>-mutated complex karyotype AML sample. The data suggest that the combined inhibition of FAK and BCL-2 enhances antileukemia activity in AML at least in part by suppressing MCL-1 and BCL-XL and that this combination may be effective in AML with <i>TP53</i> and other mutations, and thus benefit patients with high-risk AML.</p></div>

Background: Venetoclax (VEN), a B-cell lymphoma-2 (BCL-2) inhibitor, is an important drug in acute myeloid leukemia (AML) treatment regimens. However, resistance to VEN-based therapy is a common challenge in AML management. Some factors contributing to resistance include dysregulation of BCL-2 family proteins, mutations in the TP53 gene, activating kinase mutations, and oxidative phosphorylation activity. Despite these insights, other mechanisms of resistance remain elusive. We aim to analyze tumor gene expression patterns associated with treatment response in patients who received VEN-based therapy. Methods: Molecular and clinical data of the patients were obtained as part of the Oncology Research Information Exchange Network (ORIEN) Avatar dataset. RNA-Seq data from diagnostic Bone Marrow were processed to remove batch effects, and differential expression analysis was performed using DESeq2, while correcting for age at sample collection and sex of patients. Results: We included 16 individuals treated with VEN-based therapy. Median age at the time of specimen collection was 65 years (interquartile range, 58-69), 9 (56%) male, 11 (69%) had active disease despite treatment with VEN. VEN-based therapy was used as 1st line, 2nd line, 3rd line, 4th line or more in 3, 4, 4, 2, and 3 patients, respectively. We found a total of 332 genes differentially expressed (fdr<0.05 and abs(log2FoldChange)>2.0) among patients having no evidence of active disease and those with no active disease at the time of last contact. Among those genes, we found HOXA7 and PRAME of particular interest. HOXA7 has been previously implicated in Bromodomain and Extraterminal Domain Inhibitors (BETi) inhibitor resistance in AML. This makes HOXA7 a potentially universal mechanism of chemotherapy resistance in AML. PRAME has been associated with leukemia cell survival, and its knockdown has been shown to induce apoptosis in K562 cells, which is only expressed in AML cells but not in normal hematopoietic cells. Pathway analysis found that the top two enriched pathways for the differentially expressed genes were MET activates PTK2 signaling (R-HSA-8874081) and Metabolism of Angiotensinogen to Angiotensins(R-HSA-2022377). Conclusions: This study highlights some molecular mechanisms that give rise to AML resistance to VEN. Our results indicate that HOXA7 may potentially be a universal mechanism of chemotherapy resistance not limited to VEN-based therapy. Similarly, pathways enriched in resistant cells could be targeted to prevent or treat resistance to VEN-based therapy. These and other findings may help us identify resistance mechanisms in all AML cases vs those specific to VEN-based therapy, allowing the development of treatment strategies based on treatment mechanism. Also, analysis of these mechanisms can aid therapeutic decision-making and the development of effective treatment strategies to improve outcomes in AML. Citation Format: Juan Antonio Raygoza Garay, Seongseok Yun, Afaf Osman, Reshma Ramlal, David Feith, Michelle Churchman, Prajwal Dhakal. Tumor gene expression patterns affecting response to BCl-2 inhibitor venetoclax in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5852.

Venetoclax along with hypomethylating agents (HMAs) is the new standard therapy for older patients with acute myeloid leukemia (AML) not fit for intensive frontline induction chemotherapy. Venetoclax is associated with fatal episodes of tumor lysis syndrome (TLS) in chronic lymphocytic leukemia (CLL), and recommendations are for its initiation for CLL and AML in the inpatient setting with close monitoring. Herein, we evaluated the safety of outpatient venetoclax ramp up when given in addition to HMAs for the treatment of AML. We conducted a retrospective review of patients diagnosed with AML at our institution from 12/1/2016 until 7/1/2020. We identified patients who received HMAs and venetoclax for AML, either as frontline or relapsed/refractory therapy. Records were reviewed for evidence of laboratory or clinical tumor lysis episodes in all patients. Between 12/1/2016 and 7/1/2020 43, patients at our institution received venetoclax/HMA for the treatment of AML. Thirty-nine patients (91%) had venetoclax initiation and ramp up in the outpatient setting. One episode of laboratory TLS (2.5%) was identified. This patient required admission to the hospital for rasburicase and IV fluids with resolution of the laboratory effects without resultant clinical TLS. There were no episodes of clinical TLS in either group. Thirty-day mortality from venetoclax initiation was 0% in both groups. Our experience with HMAs and venetoclax showed that outpatient ramp up of venetoclax is safe with a very low risk of laboratory TLS (2.5%) and no evidence of clinical TLS within our cohort.

Novel Genetic Mechanism of Venetoclax Resistance in AML: BAX Deletion

Outcomes of De Novo Acute Myeloid Leukemia with Monocytic Differentiation (FAB M4/5) Treated with Venetoclax and Decitabine

Venetoclax synergizes with gilteritinib in FLT3 wild-type high-risk acute myeloid leukemia by suppressing MCL-1

Pharmacological inhibition of lysine specific demethylase 1 (LSD1) has shown preclinical promise as a therapy for patients with acute myeloid leukemia (AML). Numerous studies have shown that LSD1 inhibitors differentiate AML cells, decreasing the number of blast and leukemia stem cells, leading to therapeutic efficacy. With the recent clinical success of the BCL-2 inhibitor venetoclax in AML, the landscape of unmet needs for AML patients has changed. The high response rate of AML patients receiving venetoclax in combination with azacitidine as first line AML therapy means differentiation therapy with LSD1 inhibitors would be more acceptable either in combination with venetoclax, or post-venetoclax treatment. Here we explore the effects of combining the LSD1 inhibitor INCB059872 with venetoclax in AML lines that are sensitive and resistant to venetoclax. First, to test for benefits from combining venetoclax with INCB059872, we performed in vivo studies using several AML models, and each showed that combination of venetoclax and INCB059872 resulted in additive to more-than-additive efficacy when compared to either agent alone. Next, to test the effects of INCB059872 in a post-venetoclax AML setting, we generated MV4;11 and Molm13 venetoclax resistant cells by culturing them in increasing concentrations of venetoclax. These cells had elevated MCL1 expression and decreased sensitivity to venetoclax in cell proliferation assays. Treating venetoclax-resistant cells with INCB059872 alone had very little effect on their growth, while the parental cell lines were sensitive to INCB059872. However, when INCB059872 was combined with venetoclax, growth inhibition in the venetoclax resistant cell lines was apparent. Combination of INCB059872 with venetoclax appears to alter levels of the apoptotic machinery to levels that may be pro-apoptotic. In vivo studies showed no survival effects in mice engrafted with venetoclax resistant cells and treated with either venetoclax or INCB059872, but significant survival benefit was seen with the INCB059872+venetoclax combination. A third venetoclax resistant model was developed by serial transplant of murine MLL-AF9 expressing bone marrow cells from mice dosed for two weeks with 10 mg/kg QD venetoclax. After three serial transplants, resistance to venetoclax arose, while cells transplanted from vehicle treated mice were still venetoclax responsive. Venetoclax resistant MLL-AF9 driven AML was also resistant to INCB059872, but the combination of venetoclax and INCB059872 was able to slow the onset of venetoclax resistant MLL-AF9 driven AML significantly. The data presented here suggest that inhibition of LSD1 with INCB059872 may alter the expression of apoptotic machinery of AML cells leading to combinatorial therapeutic benefit when co-administered with venetoclax, and that INCB059872 combined with venetoclax may overcome acquired venetoclax resistance in AML. Citation Format: Melody Diamond, Antony Chadderton, Chunhong He, Liangxing Wu, Wenqing Yao, Holly Koblish, Sang Hyun Lee, Matthew C. Stubbs. The LSD1 inhibitor INCB059872 is a possible therapeutic option for venetoclax-resistant AML [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 1134.

6537 Background: Venetoclax is a selective inhibitor of the anti-apoptotic protein BCL2, often overexpressed in acute myeloid leukemia (AML), contributing to cell survival and resistance to standard therapies. It has expanded treatment options, especially for elderly or chemotherapy-ineligible patients. However, acquired resistance to venetoclax is a significant challenge, limiting long-term effectiveness. Understanding the mechanisms of resistance is crucial for improving therapeutic strategies and identifying biomarkers for personalized treatment. This study investigates these mechanisms by analyzing cellular phenotype, metabolic changes, BCL2 family gene/protein expression, and signaling pathways in venetoclax-resistant AML cell lines. Methods: Venetoclax-sensitive AML cell lines, MOLM-13 and MV4-11, were exposed intermittently to increasing concentrations of the drug to induce resistance. Cell viability was assessed using MTT assays, clonogenicity by colony formation, and apoptosis, mitochondrial damage, and DNA content by flow cytometry. Metabolic profiles were analyzed with Seahorse XF96, and signaling pathways were studied by Western blotting, qPCR, and global proteomics. Synergy assays were conducted with metformin (mitochondrial complex I inhibitor) and KPT-9274 (NAMPT inhibitor). Results: Intermittent exposure to venetoclax selected for resistant clones, MV4-11VR (IC 50 > 1000 nM; parental cells IC 50 = 2.5 nM) and MOLM-13VR (IC 50 = 723 nM; parental cells IC 50 = 3.3 nM). Venetoclax-induced apoptosis, mitochondrial damage, and DNA fragmentation were absent in resistant cells. Metabolic analysis showed increased mitochondrial metabolism in MOLM-13VR cells and enhanced glycolysis in MV4-11VR cells. Molecularly, MV4-11VR cells exhibited downregulation of BCL2L10, BAX, BCL2L11, BBC3, BIK, and BNIP3, while MOLM-13VR cells showed reduced BID, PMAIP1, BAD, BMF, and BECN1, along with increased MCL1. MOLM-13VR cells displayed enhanced MAPK signaling, and both resistant models had activation of the PI3K/AKT/mTOR pathway and upregulation of BCL-XL. Proteomic analysis revealed enhanced metabolic activity, with MV4-11VR cells enriched in fatty acid biosynthesis and carbohydrate metabolism pathways, while MOLM-13VR cells showed upregulation of aerobic respiration and ATP metabolism. Both parental and resistant cells exhibited comparable sensitivity to metformin and KPT-9274. The combination of these inhibitors with venetoclax resulted in synergistic effects, with KPT-9472 and venetoclax eliminating over 95% of resistant cells. Conclusions: This study provides key insights into the mechanisms of venetoclax resistance in AML. Targeting cellular metabolism with metformin or KPT-9274, combined with venetoclax, offers promising synergistic effects and potential strategies to overcome resistance, improving treatment outcomes in resistant AML cases.

Synergistic antitumor effects and mechanisms of KPT-330 combined with venetoclax in Acute Myeloid Leukemia

Background/Aims: Acute myeloid leukemia (AML) is an aggressive cancer with limited treatment options outside of chemotherapy. Improved therapies with novel mechanisms of action are desperately needed to fill this need. Both hTERT, the catalytic subunit of telomerase, and BCL-2, an apoptotic regulator, are overexpressed in AML, correlating with disease severity and poor prognosis respectively. Imetelstat is a novel, first-in-class competitive inhibitor of telomerase with clinical activity in hematologic malignancies. Venetoclax, an approved BCL-2 inhibitor for CLL, has shown a promising clinical benefit in AML patients. Preclinical evidence shows that downregulation of hTERT induces apoptosis via disruptions of hTERT and BCL-2 interaction; we hypothesize that inhibiting both targets would yield greater anti-tumor activity in AML compared to treatment with either agent alone. Methods: AML cell lines and AML patient’s PBMC samples were treated with imetelstat or venetoclax alone, or in combination, and viable and apoptotic populations of cells were evaluated by flow cytometry. Telomerase activity, hTERT expression and mitochondrial dysfunction were investigated for mechanism of action. Furthermore, an in vivo study in the MOLM-13 AML disseminated model was conducted to assess efficacy and survival. Results: A dose-dependent synergistic activity in inducing apoptosis was observed in multiple AML cell lines when combining imetelstat with venetoclax. In the MOLM-13 cell line, single-agent imetelstat and venetoclax had modest apoptotic activity after 48 hours (22% and 30% respectively), but the combination achieved 88% at 48 hours and nearly 100% at 96 hours. Similarly enhanced apoptotic activity was also observed in PBMCs purified from 4 AML patient whole blood samples. Molecular analyses showed combining imetelstat with venetoclax reduced hTERT expression and telomerase activity much more strongly than either agent alone. Furthermore, in vivo studies showed all mice tolerated the combination of imetelstat with ABT-199 well, with increased life span as compared to the vehicle control (68.1%, p=0.0001), to imetelstat (39.6%, p=0.0011) alone, or to venetoclax (23.3%, p=0.0001) alone. In the combination group, 40% of treated mice were alive 77-days after treatment discontinued whereas all mice of the other single agent arms died within two weeks, demonstrating a significant survival benefit. Conclusions: To our knowledge, this is the first investigation combining imetelstat with venetoclax in AML, and the results demonstrated a synergistic effect on induction of apoptosis in cell lines and patient samples in vitro, which translated into prolonged survival in xenograft models, thus providing a strong rationale for clinical exploration of this combination. Citation Format: Joshua J. Rusbuldt, Leopoldo Luistro, Diana Chin, Melissa Smith, Amy Wong, Margarita Romero, Aleksandra Rizo, Jacqueline Bussolari, Fei Huang, Amy (Kate) Sasser. Telomerase inhibitor imetelstat in combination with the BCL-2 inhibitor venetoclax enhances apoptosis in vitro and increases survival in vivo in acute myeloid leukemia [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 1101. doi:10.1158/1538-7445.AM2017-1101

Monosomy 7 and Del(7q) Cause Selective Sensitivity to Inhibitors of Nicotinamide Phosphoribosyltransferase in Acute Myeloid Leukemia

CD74 Antibody Conjugated to an MCL1 Inhibitor (S227928) Combines Synergistically with Venetoclax to Enhance Killing of Acute Myeloid Leukemia