BTM-3566 is a novel small-molecule compound with broad anticancer activity in hematologic and solid cancers, with exceptional activity against diffuse large B-cell lymphoma (DLBCL). The compound's mechanism of action (MoA) is dependent on activation of the ATF4 integrated stress response (ISR) by the eIF2 kinase heme-regulated inhibitor (EIF2AK1, HRI) resulting in translational repression, increased expression of ATF4, and apoptosis. To evaluate HRI's role in mediating the ISR and apoptosis by BTM-3566 in DLBCL, homozygous HRI knockout BJAB cells were generated. HRI gene deletion resulted in inhibition of BTM-3566 induction of ATF4 expression, reduction of cell growth inhibition, and abrogation of apoptosis. Expression of the antiapoptotic proteins Mcl1 and Bcl6 was reduced following treatment with BTM-3566 in wild-type BJAB cells but not in HRI KO BJAB cells. To further elucidate BTM-3566 MoA, an activity screen was performed across 407 hematologic and solid cancer cell lines. Expression of FAM210B, a mitochondrial membrane protein, emerged as a key predictor of response to BTM-3566. FAM210B expression was negatively correlated with response to compound; the cell lines most responsive to BTM-3566 were the B-cell malignancies, notably DLBCL, which have the lowest expression of FAM210B among all cancers. The relevance of FAM210B to BTM-3566 activity was confirmed by overexpression of FAM210B in the DLBCL cell line BJAB and the Burkitt lymphoma cell line RAMOS. Overexpression of FAM210B abrogated BTM-3566 antiproliferative activity in these cell lines. The specificity of BTM-3566 activity regulation by FAM210B was demonstrated by comparison to treatments known to activate the ATF4 ISR through eIF2α kinases; tunicamycin (PERK), amino acid deprivation (GCN2), ONC201, and bortezomib (HRI) were tested in FAM210B-overexpressing BJAB cells. FAM210B overexpression inhibited BTM-3566-mediated induction of ATF4 protein expression but had no effect on ATF4 induction by the other treatments. The antitumor activity of BTM-3566 in DLBCL was evaluated in patient-derived DLBCL xenograft (PDX) mouse models. Eight models (n=3/model) were tested, which included GCB and ABC subtypes, and as typical for DLBCL, all had low FAM210B expression. Treatment was initiated with BTM-3566 at 20 mg/kg po daily after tumor volume reached 200 mm3. Complete tumor regression was observed in 79% of the mice with no evidence of tumor progression during the 21-day treatment period. Treatment was well tolerated with only mild weight loss noted. In summary, BTM-3566 induces robust complete regressions in DLBCL PDX models via a unique MoA characterized by apoptosis mediated by HRI induction of the ATF4 ISR in an FAM210B-regulated manner. BTM-3566 has the potential to meaningfully address unmet clinical needs in DLBCL and other B-cell malignancies with low FAM210B expression.

Citation Format: Matthew Kostura, Jedd Levine, Alan Cooper, Andy Anantha, Michael Stocum, Michael Luther. BTM-3566 induces complete tumor regression in diffuse large B-cell lymphoma: Regulation by the eIF2α kinase “heme-regulated inhibitor” and the mitochondrial protein FAM210B [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-47.