Dan Cojocari

Background: MCL-1 is a prosurvival B-cell lymphoma 2 family protein that plays a critical role in tumor maintenance and survival and can act as a resistance factor to multiple anticancer therapies. Herein, we describe the generation and characterization of the highly potent and selective MCL-1 inhibitor ABBV-467 and present findings from a first-in-human trial that included patients with relapsed/refractory multiple myeloma (NCT04178902). Methods: Binding of ABBV-467 to human MCL-1 was assessed in multiple cell lines. The ability of ABBV-467 to induce tumor growth inhibition was investigated in xenograft models of human multiple myeloma and acute myelogenous leukemia. The first-in-human study was a multicenter, open-label, dose-escalation study assessing safety, pharmacokinetics, and efficacy of ABBV-467 monotherapy. Results: Here we show that administration of ABBV-467 to MCL-1-dependent tumor cell lines triggers rapid and mechanism-based apoptosis. In vivo, intermittent dosing of ABBV-467 as monotherapy or in combination with venetoclax inhibits the growth of xenografts from human hematologic cancers. Results from a clinical trial evaluating ABBV-467 in patients with multiple myeloma based on these preclinical data indicate that treatment with ABBV-467 can result in disease control (seen in 1 patient), but may also cause increases in cardiac troponin levels in the plasma in some patients (seen in 4 of 8 patients), without other corresponding cardiac findings. Conclusions: The selectivity of ABBV-467 suggests that treatment-induced troponin release is a consequence of MCL-1 inhibition and therefore may represent a class effect of MCL-1 inhibitors in human patients.

Purpose:Patients with acute myeloid leukemia (AML) frequently do not respond to conventional therapies. Leukemic cell survival and treatment resistance have been attributed to the overexpression of B-cell lymphoma 2 (BCL-2) and aberrant DNA hypermethylation. In a phase Ib study in elderly patients with AML, combining the BCL-2 selective inhibitor venetoclax with hypomethylating agents 5-azacitidine (5-Aza) or decitabine resulted in 67% overall response rate; however, the underlying mechanism for this activity is unknown.Experimental Design:We studied the consequences of combining two therapeutic agents, venetoclax and 5-Aza, in AML preclinical models and primary patient samples. We measured expression changes in the integrated stress response (ISR) and the BCL-2 family by Western blot and qPCR. Subsequently, we engineered PMAIP1 (NOXA)- and BBC3 (PUMA)-deficient AML cell lines using CRISPR-Cas9 methods to understand their respective roles in driving the venetoclax/5-Aza combinatorial activity.Results:In this study, we demonstrate that venetoclax and 5-Aza act synergistically to kill AML cells in vitro and display combinatorial antitumor activity in vivo. We uncover a novel nonepigenetic mechanism for 5-Aza–induced apoptosis in AML cells through transcriptional induction of the proapoptotic BH3-only protein NOXA. This induction occurred within hours of treatment and was mediated by the ISR pathway. NOXA was detected in complex with antiapoptotic proteins, suggesting that 5-Aza may be “priming” the AML cells for venetoclax-induced apoptosis. PMAIP1 knockout confirmed its major role in driving venetoclax and 5-Aza synergy.Conclusions:These data provide a novel nonepigenetic mechanism of action for 5-Aza and its combinatorial activity with venetoclax through the ISR-mediated induction of PMAIP1.

Purpose:Patients with acute myeloid leukemia (AML) frequently do not respond to conventional therapies. Leukemic cell survival and treatment resistance have been attributed to the overexpression of B-cell lymphoma 2 (BCL-2) and aberrant DNA hypermethylation. In a phase Ib study in elderly patients with AML, combining the BCL-2 selective inhibitor venetoclax with hypomethylating agents 5-azacitidine (5-Aza) or decitabine resulted in 67% overall response rate; however, the underlying mechanism for this activity is unknown.Experimental Design:We studied the consequences of combining two therapeutic agents, venetoclax and 5-Aza, in AML preclinical models and primary patient samples. We measured expression changes in the integrated stress response (ISR) and the BCL-2 family by Western blot and qPCR. Subsequently, we engineered PMAIP1 (NOXA)- and BBC3 (PUMA)-deficient AML cell lines using CRISPR-Cas9 methods to understand their respective roles in driving the venetoclax/5-Aza combinatorial activity.Results:In this study, we demonstrate that venetoclax and 5-Aza act synergistically to kill AML cells in vitro and display combinatorial antitumor activity in vivo. We uncover a novel nonepigenetic mechanism for 5-Aza–induced apoptosis in AML cells through transcriptional induction of the proapoptotic BH3-only protein NOXA. This induction occurred within hours of treatment and was mediated by the ISR pathway. NOXA was detected in complex with antiapoptotic proteins, suggesting that 5-Aza may be “priming” the AML cells for venetoclax-induced apoptosis. PMAIP1 knockout confirmed its major role in driving venetoclax and 5-Aza synergy.Conclusions:These data provide a novel nonepigenetic mechanism of action for 5-Aza and its combinatorial activity with venetoclax through the ISR-mediated induction of PMAIP1.

Hypoxia is present in most solid tumours and has been clinically correlated with poor prognosis, aggressive disease, and resistance to therapy in multiple cancer including pancreatic ductal adenocarcinoma (PDAC). It has been shown PDAC hypoxia levels are highly heterogeneous and that patient-derived-xenografts (PDXs) of PDAC have similar histological phenotypes including hypoxia to their matching primary tumours. This suggests a strong genetic determinant may underlie variations in tumour hypoxia and it is not simply the result of random events of angiogenesis. We hypothesize the steady state levels of hypoxia across patient tumours is also influenced by tumour specific differences in oxygen metabolism and tolerance to hypoxia. Genetic driven changes in cellular metabolism influence the demand for oxygen, which defines the levels and steepness of hypoxia gradients around perfused vessels. Tolerance to hypoxia determines the time cells can survive in oxygen depleted microenvironments. Adaptive hypoxia stress responses such as the activation of HIF, UPR, and autophagy pathways can affect both these factors. To investigate relationship of the two factors to hypoxia, we established a matched panel of primary PDAC, PDX, and patient-derived-organoid (PDO) models covering the clinical spectrum of hypoxia. We characterized oxygen consumption and glycolytic rates of PDOs using Seahorse XF96. Hypoxia tolerance was measured by assessing PDO regrowth characteristics under defined levels of oxygenation. We then analyzed hypoxia gradients in matching PDXs by measuring the staining of the hypoxia marker, pimonidazole, as a function of distance to the nearest perfused blood vessels with an immunofluorescence image analysis pipeline. These data allow for characterizing the degree which tumour perfusion, oxygen consumption, and hypoxia tolerance correlates with and drives hypoxia levels. As a proof of concept in targeting the two proposed factors, we investigated in PDOs the effect of inhibiting ULK1, a kinase critical to autophagy initiation downstream of the PERK/UPR pathway. The upregulation of ULK1 under hypoxia promotes survival through mitophagy and ER-phagy. This reduces cellular stress and severity of hypoxia by lowering oxygen consumption and ROS levels. Inhibition of ULK1 sensitized our panel of PDOs to severe hypoxia but at varying degrees. This is correlated with differences in their functional characteristics and genomic features. Understanding the impact of oxygen consumption and hypoxia tolerance on the individual tumour hypoxia levels sets the stage for identifying genetic drivers of tumour hypoxia and development of hypoxia-targeted therapies. Citation Format: Ji Zhang, Dan Cojocari, Pedro Boasquevisque, Mark Zaidi, Trevor McKee, Nikolina Radulovich, Ming-Sound Tsao, David Hedley, Marianne Koritzinsky, Bradley Wouters. Therapeutic targeting of hypoxia tolerance and oxygen consumption in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 117.

Purpose: Patients with acute myeloid leukemia (AML) frequently do not respond to conventional therapies. Leukemic cell survival and treatment resistance have been attributed to the overexpression of B-cell lymphoma 2 (BCL-2) and aberrant DNA hypermethylation. In a phase Ib study in elderly patients with AML, combining the BCL-2 selective inhibitor venetoclax with hypomethylating agents 5-azacitidine (5-Aza) or decitabine resulted in 67% overall response rate; however, the underlying mechanism for this activity is unknown. Experimental Design: We studied the consequences of combining two therapeutic agents, venetoclax and 5-Aza, in AML preclinical models and primary patient samples. We measured expression changes in the integrated stress response (ISR) and the BCL-2 family by Western blot and qPCR. Subsequently, we engineered PMAIP1 (NOXA)- and BBC3 (PUMA)-deficient AML cell lines using CRISPR-Cas9 methods to understand their respective roles in driving the venetoclax/5-Aza combinatorial activity. Results: In this study, we demonstrate that venetoclax and 5-Aza act synergistically to kill AML cells in vitro and display combinatorial antitumor activity in vivo. We uncover a novel nonepigenetic mechanism for 5-Aza–induced apoptosis in AML cells through transcriptional induction of the proapoptotic BH3-only protein NOXA. This induction occurred within hours of treatment and was mediated by the ISR pathway. NOXA was detected in complex with antiapoptotic proteins, suggesting that 5-Aza may be “priming” the AML cells for venetoclax-induced apoptosis. PMAIP1 knockout confirmed its major role in driving venetoclax and 5-Aza synergy. Conclusions: These data provide a novel nonepigenetic mechanism of action for 5-Aza and its combinatorial activity with venetoclax through the ISR-mediated induction of PMAIP1.