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Supplementary Table 1: Table provides information on the siRNA sequences and/or chemical structures of the non-FDA approved inhibitors for bromodomain proteins, transcription factors and kinases described in the current study. Supplementary Table 2: Sequences of primers used for qRT-PCR analysis of various targets described in the study. Supplementary Table 3: Sequences of primers used for ChIP analysis of various targets described in the study. Supplementary Table 4: Analysis of HOXB13 target gene expression in the GSE92721 data set. Supplementary Table 5: Analysis of HOTBIN10 gene expression in the GSE92721data set. Supplementary Table 6: Analysis of HOTBIN10 gene expression in the Moffitt TCC data set. Supplementary Table 7: Clinical parameters of the primary cancers in the Moffitt TCC data set. Supplementary Table 8: Clinical parameters of the metastatic cancers in the Moffitt TCC data set. Supplementary Table 9: Analysis of HOTBIN10 genes in MSKCC gene expression data set (GSE21034). Supplementary Table 10: Analysis of HOTBIN10 gene expression in the GSE67980 CTC data set.

Supplementary Fig. 1. Epigenetic landscape and the transcription cofactor recruitment at the HOXB13 enhancer region under various treatments; Supplementary Figure 2. BRD4 is a target of the dual BET-kinase inhibitors in CRPCs; Supplementary Figure 3. Sensitivity of normal and AR negative cell lines to the novel BET-kinase inhibitors; Supplementary Figure 4. HOXB13 but not FOXA1 is under the epigenetic control of BRD4 in prostate cancer cells; Supplementary Figure S5. Haploinsufficiency of HOXB13 sensitizes CRPCs to the novel BET-kinase inhibitors; Supplementary Figure S6. AURKB expression correlates with HOXB13 in human CTCs from mCRPC patients following Abiraterone therapy; Supplementary Fig. S7. AURKB is a therapeutic vulnerability of HOXB13 positive prostate cancers

Multiple myeloma (MM) is an incurable disease. Classical chemotherapeutics including bortezomib, melphalan, lenalidomide and thalidomide have greatly enhanced survival times. Unfortunately, patients typically relapse and become refractory with an average survival of 5 years post-diagnosis. Our emerging studies demonstrate a novel role for ULK3 in regulating autophagy in MM, a key program that sustains cell survival under times of stress and has been implicated as a major mechanism of proteasome inhibitor (PI) resistance. MM is known to be highly dependent on autophagy and, currently, specific ULK3 inhibitors are lacking. We posit that by targeting this marker in chemotherapy resistant MM patients, we can circumvent alternative metabolic routes and resensitize to standard of care proapoptotic therapy. We performed RNASeq analysis of CD138+ MM cells derived from patients across the disease stages spectrum (n=815) to confirm the role of ULK3 in disease progression and resistance to chemotherapy. We developed novel inhibitors SG3014/MA9060 that target multiple kinases including ULK3 (EC50 90nM) as well as BRD4. BRD4 is a known driver of MYC and its expression is increased in refractory MM. The BRD4 inhibitor, JQ1, effectively impairs the tumorigenic potential of MM but resistance has also been noted. We determined the efficacy of MA9060 for the treatment of CD138+ MM isolated from naive and refractory patients using a novel ex vivo high throughput platform developed at Moffitt.ULK3 is highly associated with MM stage of the disease. Refractory MM patients have increased autophagy activity with significantly higher expression of ULK3 in refractory patients and in drug resistant cell lines (immunoblotting U266 vs U266-PSR; RPMI-8226 vs RPMI-8226-B25; ABNL vs V10 resistant cells).Genetic ablation of ULK3 by siRNA in U266 and 8226 cell lines results in rapid cessation of the downstream autophagy proteins (ULK1, ATG13, pATG13) and MM cell death within 72h of transduction. Increased concentrations of autophagy inhibitors MA9060/SG3014 progressively decreased CMYC and ULK3 levels, as measured by immunoblotting in U266 cells. In vivo preclinical model of U266Luc tail vein injection proved our drugs are highly effective in reducing tumor dissemination and extending overall survival (CTRL untreated n=65 days vs MA9060 n=110). Importantly, we noted no overt toxicity and protected effect against myeloma-induced bone disease. This novel class of drug works synergistically with PI and can re-sensitize PI resistant disease to these effective therapies. We also show by EMMA ex vivo platform that MA9060 is highly effective for the treatment of CD138+ MM cells isolated from patients with refractory disease.ULK3 represents a novel target for treatment of MM refractory disease. Our dual inhibitors can increase overall survival in vivo and ex vivo, therefore we expect to quickly translate our novel molecules to the clinic. Citation Format: Marilena Tauro, Tao Li, Mark Meads, Praneeth R. Sudalagunta, Raghunandan R. Alugubelli, Nicholas J. Lawrence, Ernst Schonbrunn, Harshani Lawrence, Kenneth H. Shain, Conor C. Lynch. Novel autophagy inhibitory strategies to overcome chemotherapy resistance in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 452.

Gene expression is regulated by promoters and enhancers marked by histone H3 lysine 27 acetylation (H3K27ac), which is established by the paralogous, multidomain-containing histone acetyltransferases (HAT) EP300 and CBP. These enzymes display overlapping regulatory roles in untransformed cells, but less characterized roles in cancer cells. We demonstrate that the majority of high-risk pediatric neuroblastoma (NB) depends on EP300, whereas CBP has a limited role. To disrupt EP300, we developed a proteolysis-targeting chimera (PROTAC) compound termed “JQAD1” that selectively targets EP300 for degradation. JQAD1 treatment causes loss of H3K27ac at the enhancers that regulate NB master transcription factors, and drives rapid NB apoptosis, with limited toxicity to untransformed cells where CBP may compensate. In parallel, we demonstrate that EP300 and CBP have subdomain-specific functions, with enriched activity for individual domains in distinct tumor lineages. These data provide a foundation for interrogation of these histone acetyltransferases in distinct tumor types and new strategies for therapeutic disruption of subdomain and scaffolding activities of these multidomain proteins in cancer. Citation Format: Adam D. Durbin, Noha Shendy, Audrey Mercier, Yang Zhang, Melissa J. Bikowitz, Logan H. Sigua, Sarah Robinson, Tingjian Wang, Barbara Jonchere, A. Thomas Look, Mark W. Zimmerman, Martine Roussel, Brian J. Abraham, Ernst Schonbrunn, Kimberly Stegmaier, Jun Qi. Targeting EP300 and CBP for therapeutic benefit in pediatric solid tumors. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR005.