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Figure S2. ABBV-744 retains strong transcription regulatory activities in AML cells and it did not alter the baseline expression of representative IFN-r or PMA responsive genes.

MCL-1 is known to play a major role in resistance to BCL-2 inhibition, but the contribution of other BCL-2 family proteins has not been fully explored. We, here, demonstrate the ineffectiveness of MCL-1 inhibitor AMG176 in venetoclax-resistant, and conversely, of venetoclax in AMG176-resistant acute myelogenous leukemia (AML). Like cells with acquired resistance to venetoclax, cells with acquired resistance to AMG176 express increased MCL-1. Both cells with acquired resistance to venetoclax and to AMG176 express increased levels of BCL-2 and BCL-2A1, decreased BAX, and/or altered levels of other BCL-2 proteins. Cotargeting BCL-2 and MCL-1 was highly synergistic in AML cell lines with intrinsic or acquired resistance to BH3 mimetics or engineered to genetically overexpress BCL-2 or BCL-2A1 or downregulate BAX. The combination effectively eliminated primary AML blasts and stem/progenitor cells resistant to or relapsed after venetoclax-based therapy irrespective of mutations and cytogenetic abnormalities. Venetoclax and AMG176 combination markedly suppressed antiapoptotic BCL-2 proteins and AML stem/progenitor cells and dramatically extended mouse survival (median 336 vs. control 126 days; P < 0.0001) in a patient-derived xenograft (PDX) model developed from a venetoclax/hypomethylating agent therapy-resistant patient with AML. However, decreased BAX levels in the bone marrow residual leukemia cells after 4-week combination treatment may represent a resistance mechanism that contributed to their survival. Enhanced antileukemia activity was also observed in a PDX model of monocytic AML, known to be resistant to venetoclax therapy. Our results support codependence on multiple antiapoptotic BCL-2 proteins and suppression of BAX as mechanisms of AML resistance to individual BH3 mimetics. Cotargeting of MCL-1 and BCL-2 eliminates otherwise apoptosis-resistant cells.

Figure S1.ABBV-744 exhibits potent anti-proliferative activity against AML cells through cell cycle arrest and induction of apoptosis.

Figure S5.Antitumor activity of ABBV-744/venetoclax in AML xenograft model and AML primary samples

Primary patient sample information

Supplementary Figure from Maximal Activation of Apoptosis Signaling by Cotargeting Antiapoptotic Proteins in BH3 Mimetic–Resistant AML and AML Stem Cells

Supplementary Data from Maximal Activation of Apoptosis Signaling by Cotargeting Antiapoptotic Proteins in BH3 Mimetic–Resistant AML and AML Stem Cells

Supplementary figure and table legends

Tumor growth inhibition in cell line-based xenograft models and supporting statistics.

Dual bromodomain BET inhibitors that bind with similar affinities to the first and second bromodomains across BRD2, BRD3, BRD4, and BRDT have displayed modest activity as monotherapy in clinical trials. Thrombocytopenia, closely followed by symptoms characteristic of gastrointestinal toxicity, have presented as dose-limiting adverse events that may have prevented escalation to higher dose levels required for more robust efficacy. ABBV-744 is a highly selective inhibitor for the second bromodomain of the four BET family proteins. In contrast to the broad antiproliferative activities observed with dual bromodomain BET inhibitors, ABBV-744 displayed significant antiproliferative activities largely although not exclusively in cancer cell lines derived from acute myeloid leukemia and androgen receptor positive prostate cancer. Studies in acute myeloid leukemia xenograft models demonstrated antitumor efficacy for ABBV-744 that was comparable with the pan-BET inhibitor ABBV-075 but with an improved therapeutic index. Enhanced antitumor efficacy was also observed with the combination of ABBV-744 and the BCL-2 inhibitor, venetoclax compared with monotherapies of either agent alone. These results collectively support the clinical evaluation of ABBV-744 in AML (Clinical Trials.gov identifier: NCT03360006).

Figure S3.Comparison of ABBV-744 and GSK046 BD2 selective compounds.

Figure S6.Identification of biomarker of response to ABBV-744/venetoclax therapy.

Figure S4.ABBV-744 displaces BRD4 from regulatory regions of BCL2, BCL2L1 and RUNX1 in sensitive AML cells but not insensitive AML line.

Acute myeloid leukemia (AML) stem cells (AMLSCs) are thought to be responsible for disease initiation, chemoresistance and treatment failure. AMLSCs and residual cytarabine (AraC)-resistant AML cells (constituting minimal residual disease, MRD) were shown to be highly dependent on mitochondrial function for survival and thus are vulnerable to pharmacological blockade of the oxidative phosphorylation (OXPHOS) (Farge et al. Cancer Discov, 2017). Efficacy of OXPHOS inhibitor IACS-010759 was previously reported, demonstrating potent inhibition of mitochondrial complex I, OXPHOS suppression and growth inhibition of AML cells (Molina, et al. Nat Med, 2018). Here we evaluated OXPHOS dependency of AML MRD cells and determined impact of OXPHOS blockade on residual AML cells surviving standard chemotherapy (Doxorubicin/AraC, DA). Our results demonstrated that AML cell lines treated with AraC or DA induced accumulation of reactive oxygen species, mitochondrial superoxides, increased mitochondrial mass and mitochondrial membrane potential. AraC- and DA-based therapies in vitro were significantly potentiated by IACS-010759. OxPhos dependency shown as a significantly increased basal and maximal oxygen consumption rate after AraC and DA treatment, was fully inhibited by OxPhos inhibitor, leading to a total mitochondrial collapse. OxPhos inhibition in combination with DA consolidated reduction of viable cell number, induction of apoptosis and differentiation in cell lines and in AraC-resistant primary patient samples. Next, the efficacy of IACS-010759 together with DA chemotherapy was evaluated in several chemotherapy-sensitive and -resistant animal models in vivo. DA/IACS-010759 combination significantly reduced leukemia burden and significantly extended survival in OCI-AML3/Luc/GFP model and in FLT3-ITD+ AML PDX model. In the latter, IACS-010759 led to reduction of leukemia burden, and delayed leukemia recurrence when administered post completion of DA. At the single-cell level, CyTOF analysis demonstrated that this combination reduced frequency of CD34+CD38lowCD123+AML LSCs and has superior efficacy to facilitate differentiation of immature subpopulations. Furthermore, addition of IACS-010759 to DA extended survival of mice inoculated with chemoresistant PDX AML models. Finally, IACS-010759 administered during consolidation phase significantly extended mice survival compared to standard of care arm, supporting clinical utility of OXPHOS inhibitors in AML. In conclusion, our findings indicate that chemotherapy fosters mitochondrial respiration in AML, which could be abrogated by OXPHOS inhibitor at the LSCs and MRD level, in vitro and in vivo. Our data advocate for combining mitochondrial targeting strategies with chemotherapy as a part of induction and consolidation treatment for improved control of MRD, eradication of AMLSC and extended response duration. Citation Format: Natalia Baran, Lina Han, Lucille Stuani, Antonio Cavazos, Laurie M. Cooper, Cassandra L. Ramage, Vinitha M. Kuruvilla, Qi Zhang, Marie Sabatier, Emeline Boet, Jason P. Gay, Ningping Feng, Lokesh Battula, Emeline Chu-Van, Florence Castelli, Martin Carrol, Sergej Konoplev, Beenu Thakral, Hagop M. Kantarjian, Naval Daver, Joseph Marszalek, Michael Andreeff, Jean-Emmanuel Sarry, Marina Konopleva. Targeting mitochondrial metabolism following Induction Chemotherapy eradicates Acute Myeloid Leukemia at the level of Minimal Residual Disease [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A24.

MCL-1 is known to play a major role in resistance to BCL-2 inhibition, but the contribution of other BCL-2 family proteins has not been fully explored. We, here, demonstrate the ineffectiveness of MCL-1 inhibitor AMG176 in venetoclax-resistant, and conversely, of venetoclax in AMG176-resistant acute myelogenous leukemia (AML). Like cells with acquired resistance to venetoclax, cells with acquired resistance to AMG176 express increased MCL-1. Both cells with acquired resistance to venetoclax and to AMG176 express increased levels of BCL-2 and BCL-2A1, decreased BAX, and/or altered levels of other BCL-2 proteins. Cotargeting BCL-2 and MCL-1 was highly synergistic in AML cell lines with intrinsic or acquired resistance to BH3 mimetics or engineered to genetically overexpress BCL-2 or BCL-2A1 or downregulate BAX. The combination effectively eliminated primary AML blasts and stem/progenitor cells resistant to or relapsed after venetoclax-based therapy irrespective of mutations and cytogenetic abnormalities. Venetoclax and AMG176 combination markedly suppressed antiapoptotic BCL-2 proteins and AML stem/progenitor cells and dramatically extended mouse survival (median 336 vs. control 126 days; P < 0.0001) in a patient-derived xenograft (PDX) model developed from a venetoclax/hypomethylating agent therapy-resistant patient with AML. However, decreased BAX levels in the bone marrow residual leukemia cells after 4-week combination treatment may represent a resistance mechanism that contributed to their survival. Enhanced antileukemia activity was also observed in a PDX model of monocytic AML, known to be resistant to venetoclax therapy. Our results support codependence on multiple antiapoptotic BCL-2 proteins and suppression of BAX as mechanisms of AML resistance to individual BH3 mimetics. Cotargeting of MCL-1 and BCL-2 eliminates otherwise apoptosis-resistant cells.

BCL-XL is a well-validated cancer target. However, the on-target and dose-limiting thrombocytopenia limits the use of BCL-XL inhibitors such as ABT263 as safe and effective anticancer agents. To reduce the toxicity of ABT263, we converted it into DT2216, a BCL-XL proteolysis targeting chimera (PROTAC), that targets BCL-XL to the Von Hippel-Lindau (VHL) E3 ligase for degradation. We found that DT2216 was more potent against various BCL-XL-dependent leukemia and cancer cells but significantly less toxic to platelets than ABT263 in vitro because VHL is poorly expressed in platelets. In vivo, DT2216 effectively inhibits the growth of several xenograft tumors as a single agent or in combination with other chemotherapeutic agents, without causing significant thrombocytopenia. These findings demonstrate the potential to use PROTAC technology to reduce on-target drug toxicities and rescue the therapeutic potential of previously undruggable targets. Furthermore, DT2216 may be developed as a safe first-in-class anticancer agent targeting BCL-XL.