MCL-1 and PKA/AMPK Axis Fuel Venetoclax Resistance in Lymphoid Cancers

Abstract

Venetoclax, the first approved BH3 mimetic targeting BCL2, demonstrates high response rate in chronic lymphocytic leukemia (CLL) but resistant cases are emerging. Aside from BCL2 mutations affecting venetoclax binding, multiple lines of mounting evidence suggest a role for non-mutational mechanisms underlying resistance to this drug. By applying both CRISPR-Cas9 knock-out and ORF overexpression screens in the lymphoma cell line OCI-Ly1, we previously reported the identification of MCL-1 overexpression and of the AMPK/PKA signaling axis in altering energy metabolism underlying venetoclax resistance (Guieze, ASH 2018). Here, we report further in-depth exploration of the impact of these findings, discovered through the analysis of lymphoid cell lines, and of specimens collected from CLL patients developing venetoclax resistance. The resistant lymphoma cell lines that we generated (OCI-Ly1 and SU-DHL4 cells) displayed increased oxidative phosphorylation (OXPHOS) compared to the parental lines, measured by Seahorse assay. We instead observed that venetoclax rapidly perturbs OXPHOS in sensitive cells. This process is dependent on mitochondrial outer membrane permeabilization, as this effect is abrogated in BAX/BAK1 double knockout (KO) cells. Targeting OXPHOS was shown to synergize with venetoclax in vitro and in vivo, as combination of venetoclax and oligomicin (an inhibitor of the ATP synthase, the complex V of the mitochondrial electron transport chain), was more effective than each drug alone in reducing tumor growth of a subcutaneous NSG xenograft model based on OCI-Ly1. Among the candidate markers driving resistance identified from the genome-wide screens, we focused on AMP pathway members (AMPK and PKA) and the ID3 transcriptional regulator, given that ID3 KO cells demonstrated similar transcriptomic changes than the resistant OCI-Ly1 cells. We found that PRKAR2B (encoding a PKA subunit), already highlighted in our ORF screen, was the top transcript overexpressed when knocking out ID3. To clarify how the dominant-negative transcription factor ID3 regulates PRKAR2B expression, we performed ATAC-seq of the ID3 OCI-Ly1 knockout (vs control) lines in order to determine differential signatures of chromatin accessibility and transcription factor engagement. We showed that ID3 repression leads to genome-wide increased accessibility associated with motifs of the lymphoid transcription factor TCF3. TCF3 has previously been shown to interact with ID3 and to be involved in the transcription of ADIPOQ, which was identified in the GOF screen. TCF3 binding sites were confirmed to be present within putative enhancer regions of PRKAR2B in a B cell context. We then investigated whether our findings could be validated in patient samples. By whole-exome sequencing of matched pretreatment and venetoclax-resistant CLL samples collected from 6 patients, we did not detect any recurrent somatic mutations associated with resistance. The resistant samples from three of 6 patients, however, harbored subclones with 1q amplification in a common region encompassing the MCL1 locus. We identified 4 additional CLL cases relapsing on venetoclax with leukemia samples collected before and after relapse. By immunohistochemical staining of 9 of 10 cases for which tissue was available, we detected increased MCL-1 expression at relapse in 6 of 9 cases (p = 0.026). We furthermore confirmed the involvement of AMPK signaling by detecting evidence of AMPK, ACC and p-ACC expression in 4 of 9 patients (all p = 0.0062). ID3 expression was decreased at matched relapse samples (p = 0.0001), supporting the presence of the resistance circuit we identified above. Taken together, our results identified the increased MCL-1 expression and PKA/AMPK activation as underlying mechanisms for venetoclax resistance. Our data support the implementation of combinatorial therapy with metabolic modulators to address venetoclax resistance. Disclosures Guièze: Abbvie: Honoraria; Roche: Honoraria; Janssen: Honoraria; Gilead: Honoraria. Thompson:AbbVie: Research Funding; Amgen: Consultancy, Research Funding; Pfizer: Research Funding; Pharmacyclics: Research Funding; Genentech: Consultancy, Honoraria; Gilead: Consultancy, Honoraria. Davids:AbbVie, Acerta Pharma, Adaptive, Biotechnologies, Astra-Zeneca, Genentech, Gilead Sciences, Janssen, Pharmacyclics, TG therapeutics: Membership on an entity's Board of Directors or advisory committees; Research to Practice: Honoraria; AbbVie, Astra-Zeneca, Genentech, Janssen, MEI, Pharmacyclics, Syros Pharmaceuticals, Verastem: Consultancy; Acerta Pharma, Ascentage Pharma, Genentech, MEI pharma, Pharmacyclics, Surface Oncology, TG Therapeutics, Verastem: Research Funding. Brown:AbbVie: Consultancy; Acerta Pharma: Consultancy; Loxo: Consultancy, Research Funding; BeiGene: Consultancy; Catapult Therapeutics: Consultancy; AstraZeneca: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Pharmacyclics: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy, Research Funding; Sun Pharmaceuticals: Research Funding; Janssen: Honoraria; Teva: Honoraria; Morphosys: Other: Data safety monitoring board; Invectys: Other: Data safety monitoring board; Octapharma: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding; Juno/Celgene: Consultancy; Dynamo Therapeutics: Consultancy; Genentech/Roche: Consultancy; Gilead: Consultancy, Research Funding. Wierda:Xencor: Research Funding; Cyclcel: Research Funding; Genentech: Research Funding; Pharmacyclics LLC: Research Funding; Gilead Sciences: Research Funding; KITE pharma: Research Funding; Oncternal Therapeutics Inc.: Research Funding; Sunesis: Research Funding; AbbVie: Research Funding; Janssen: Research Funding; Acerta Pharma Inc: Research Funding; GSK/Novartis: Research Funding; Miragen: Research Funding; Loxo Oncology Inc.: Research Funding; Juno Therapeutics: Research Funding. Letai:AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding; Zeno Pharmaceuticals, Vivid Bioscience, Flash Therapeutics, Dialectic Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Cofounder or Advisory Board member. Neuberg:Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Mootha:Jansen Pharmaceuticals: Other: SAB, compensation; 5am Ventures: Other: SAB, compensation; Raze Therapeutics: Other: Founder, SAB, equity. Getz:MuTect, ABSOLTUE, MutSig and POLYSOLVER: Patents & Royalties: MuTect, ABSOLTUE, MutSig and POLYSOLVER; Pharmacyclics: Research Funding; IBM: Research Funding. Wu:Pharmacyclics: Research Funding; Neon Therapeutics: Other: Member, Advisory Board.

Venetoclax resistance induced by activated T cells can be counteracted by sphingosine kinase inhibitors in chronic lymphocytic leukemia

“…While Liu et al. reported that the increase in MCL-1 expression is one important mechanisms for venetoclax resistance ( 35 ), others found that BCL-XL is a major regulator of this process in CLL ( 9 , 36 ). In line with our result, Haselager et al.…”

“…The role of MCL-1 and BCL-XL in the resistance to venetoclax is still a matter of debate. While Liu et al reported that the increase in MCL-1 expression is one important mechanisms for venetoclax resistance (35), others found that BCL-XL is a major regulator of this process in CLL (9,36). In line with our result, Haselager et al demonstrated that there is a hierarchy of BCL-2 family members in CLL cells under the pressure of venetoclax, in which BCL-XL is dominant over MCL-1 in CLL venetoclax resistance when both are present (9,36).…”

Mechanisms of venetoclax resistance and solutions

“…This data suggests there is tremendous potential of venetoclax in treating hematologic tumors (54). The finding reported by R Guièze et al that venetoclax rapidly interferes with OXPHOS in sensitive CLL cells also further suggests that venetoclax effectively targets OXPHOS in lymphoid malignancies (24) to promote apoptosis in tumor cells (54). However, the resistance of venetoclax may be caused by the upregulation of OXPHOS.…”

“…After venetoclax treatment, the changes in the relative expression of BCL-2, MCL-1, and BCL-XL, resulted in secondary drug resistance ( 22 , 23 ). Significantly elevated MCL-1 expression levels were observed in samples collected from patients who relapsed after venetoclax treatment, suggesting that it may be a mechanism to secondary drug resistance ( 24 ).…”

“…The investigators investigated the factors contributing to these changes and found that the copy number in the chromosomal 1q23 region increased in resistant cells, resulting in the upregulation of AMPK-related proteins encoded in this region, which are key molecules regulating cellular energy metabolism. This change increased OXPHOS levels, and the reduced cellular energy levels caused by venetoclax-induced cytochrome c release are replenished, thereby mediating drug resistance ( 24 , 54 ). The application of AMPK and ETC complex inhibitors to these cell lines results in increased sensitivity to venetoclax, while activators result in resistance.…”