Bantam Data in Collaboration with MD Anderson Presented at 2024 AACR Conference

The integrated stress activator BTM-3566 overcomes therapeutic resistance in mantle cell lymphoma Yang Liu1, Heng-Huan Lee1, Joseph McIntosh1, Yijing Li1, Tianci Zhang1, Wei Wang1, Matthew J Kostura2 and Michael Wang1,3 1Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2Bantam Pharmaceutical, 8 Davis Dr. Suite 220, Research Triangle Park, NC, USA 3Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA ABSTRACT CONTACT Yang Liu, PhD [email protected] Michael Wang, MD [email protected] MD Anderson Cancer Center Successive generations of BTK inhibitors and CAR T-cell therapy have transformed the treatment of mantle cell lymphoma (MCL). However, treatment resistance has inevitably emerged, creating significant medical need.1 Recently a novel, clinical stage small molecule, BTM-3566, has been described that targets a mitochondrial process essential for Diffuse Large B-cell lymphoma (DLBCL) survival. BTM- 3566 activates the mitochondrial protease OMA1, triggering the ATF4 integrated stress response (ISR).2 BTM-3566 has robust in vitro efficacy in DLBCL cell lines irrespective of genomic background and elicits complete tumor regression in multiple cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models of DLBCL. Based on the activity observed in DLBCL, we further explored the effectiveness of BTM-3566 in MCL models in vitro and in xenograft mouse models in vivo. INTRODUCTION Figure 1. BTM-3566 induces apoptosis via the ISR pathway. • The CellTiter-Glo® cell viability assay (Promega) was employed to assess cell viability following a 72-hour treatment. • Apoptosis inductive effects were measured through Annexin V/PI staining after 24h treatment. • A JeKo-1 xenograft mouse model and four PDX mouse models, derived from patients exhibiting multiple clinical resistance, were passaged and utilized for in vivo screening. BTM-3566 induced the activation of the mitochondrial protease OMA1, leading to the cleavage of DELE1 and OPA1. OPA1 activation leads to fragmentation of the mitochondrial network. DELE1 cleavage leads to activation of HRI and downstream effector pathways leading to cancer cell death. RESULTS REFERENCES • BTM-3566 inhibited cell proliferation and induced apoptosis across a panel of MCL cell lines, irrespective of resistance status to ibrutinib and venetoclax. • BTM-3566 induced dose-dependent reductions in MCL-1 and c- MYC, accompanied by an increase in the transcription factor ATF4, indicative of activation of the stress response. • BTM-3566 treatment resulted in nearly complete tumor growth inhibition in CDX and therapeutic resistant PDX mouse models. CONCLUSIONS Click here to insert your Conclusions text. Type it in or copy and paste from your Word document or other source. 1. Yijing Li et al. Potentiation of apoptosis in drug-resistant mantle cell lymphoma cells by MCL-1 inhibitor involves downregulation of inhibitor of apoptosis proteins. Cell Death & Disease. 2023, 2023, 14(11):714 2. Adrian Schwarzer et al. Targeting Aggressive B-cell Lymphomas through Pharmacological Activation of the Mitochondrial Protease OMA1. Mol Cancer Ther. 2023, 22(11):1290-1303 Figure 3. BTM-3566 induced apoptosis in MCL cell lines. * p <0.1, ** p<0.01, ***P<0.001 and ****p<0.0001 0 10 20 30 40 0 1000 2000 3000 Days post cell inoculation Tumor Volume (mm3) Vehicle BTM-3566-10mg/kg BTM-3566-20mg/kg BTM-3566-30mg/kg ✱✱ ✱✱ ✱✱ 0 10 20 30 40 0.0 0.5 1.0 1.5 Days post cell inoculation Body Weight (ratio) Vehicle BTM-3566-10mg/kg BTM-3566-20mg/kg BTM-3566-30mg/kg JeKo-1 CDX model 0 5 10 15 20 25 0 1000 2000 3000 Rituximab-R PDX Days post treatment Tumor Volume (mm3) Vehicle BTM-3566 ✱✱✱✱ 0 5 10 15 20 0 1000 2000 3000 4000 ACP-196/VEN/CAR T-R PDX Days post treatment Tumor Volume (mm3) Vehicle BTM-3566 ✱✱✱✱ 0 5 10 15 20 0 2000 4000 6000 IBN/PBN/CAR T-R PDX Days post treatment Tumor Volume (mm3) Vehicle BTM-3566 ✱✱✱✱ 0 10 20 30 0 1000 2000 3000 4000 5000 Naive PDX Days post treatment Tumor Volume (mm3) Control BTM-3566 ✱✱✱✱ METHODS AND MATERIALS Figure 4. BTM-3566 reduced MCL-1 and c-MYC expression, while increased ATF4 expression after 24h treatment. Figure 2. BTM-3566 inhibited cell growth in a panel of MCL cell lines. 0.001 0.01 0.1 1 10 100 0 50 100 Concentration (μM) Cell viability (%) BTM-3566 0 200 400 600 800 IC50 (nM) JeKo-1 JeKo BTK KD_1 JeKo BTK KD_2 JeKo-IBN-R Mino Mino-VEN-R Rec-VEN-R JVM-2 JVM-13 MAVER-1 025050010005000 025050010005000 025050010005000 025050010005000 0 20 40 60 BTM-3566 (nM) Apoptotic cells (%) JeKo-1 JeKo-IBN-R Mino Mino-VEN-R * * ** *** ns***** ** * **** **** **** * ** ** *** Figure 5. BTM-3566 inhibited tumor growth in JeKo-1 CDX model. ** p<0.01 Figure 6. BTM-3566 (20 mg/kg) inhibited tumor growth in MCL PDX mouse models. IBN: ibrutinib; PBN: pirtobrutinib; VEN: venetoclax. ****p<0.0001. 676

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