Science Spotlight

Hit the “reset” button on acquired drug resistance of cancer cells

From the Bradley Lab, Public Health Sciences and Basic Sciences Divisions and collaborating Abdel-Wahab Lab, Memorial Sloan Kettering Cancer Center

Cancer resistance to drug therapy can result from long-term use and arises through various ways, including an increase of anti-apoptotic proteins (BCL2), genetic mutations (e.g., TP53 and BAX), or altered metabolism of cells. All of these examples have been observed for acute myeloid leukemia (AML), a cancer of bone marrow cells that typically differentiate into white blood cells. Despite the development of new and effective therapies, alternative strategies to treating drug resistant AML are needed. The collaborating labs of Dr. Robert Bradley at Fred Hutch Cancer Center and Dr. Omar Abdel-Wahab at Memorial Sloan Kettering Cancer Center sought to hit the “reset” button on acquired drug resistance of AML by identifying and manipulating mechanisms of resistance. They published their findings recently in Cancer Cell.

To uncover which host genes are responsible for drug resistance to AML therapies, the researchers performed a genome-wide CRISPR screen to individually knockout host genes in AML cells, followed by treatment of cells with one of the AML therapies – venetoclax, 5-azacytidine, cytarabine, etoposide, midostaurin, or idarubicin. Analysis of these data identified two relevant groups: host genes that sensitize AML to the drug (sensitizers) and those that are associated with drug resistance (resistance) as diagrammed in the image below. Consistent with several previous reports, genes related to venetoclax resistance (BAX, PMAIP, and TP53) were among the top hits in their screen for this drug. Next, the researchers analyzed the top gene candidates to determine which functional categories of genes had the highest enrichment. For venetoclax, an inhibitor of the anti-apoptotic BCL2 protein, the researchers observed an enrichment of sensitizer genes with involvement in RNA splicing and mRNA regulation. This finding was unique to venetoclax and was not observed for the other tested AML therapies.

CRISPR-based knockout of RNA-binding proteins were completed in AML cell lines then treated with AML therapeutics to identify proteins involved in resistance and susceptibility to each therapeutic. These genes of interest were validated using genetic and pharmacological approaches. RBM10 loss or drug inhibition of certain splicing-dependent kinases resensitized AML cells to venetoclax-mediated cell death.
CRISPR-based knockout of RNA-binding proteins were completed in AML cell lines then treated with AML therapeutics to identify proteins involved in resistance and susceptibility to each therapeutic. These genes of interest were validated using genetic and pharmacological approaches. RBM10 loss or drug inhibition of certain splicing-dependent kinases resensitized AML cells to venetoclax-mediated cell death. Figure taken from primary publication

Others have published a similar correlation between RNA processing factors and tumor growth or drug resistance of leukemias. Therefore, the researchers investigated which RNA processing genes were related to either sensitizing or resistance activity in the context of venetoclax treatment. To do this, the researchers conducted a CRISPR-based screen of RNA processing genes and validated several of these factors as sensitizers to venetoclax: RBM10, HNRNPF, HNRNPD, SRSF11, and HNRNPAB. Further characterization of RBM10, a gene encoding an RNA splicing factor, showed that venetoclax-resistant cell lines had limited cell death following RBM10 knockout but secondary treatment with venetoclax induced significant cell death. These findings suggest that targeting the RBM10 splicing factor as a therapeutic strategy might be insufficient as a monotherapy and instead, it should be combined with venetoclax as a drug re-sensitizing approach. Along these same lines, their studies in mice with drug-resistant AML cell transplants demonstrated prolonged survival for those mice that received RBM10 knockout cells in combination with venetoclax treatment as compared to the control mice. Importantly, these cell-killing effects were specific to the tumor cells and did not occur for non-cancerous types of blood cells.

Lastly, the researchers linked the mechanism of RBM10-dependent venetoclax resistance to mis-splicing of XIAP, a gene also known as BIRC4 that encodes an anti-apoptotic protein. By targeting splicing factors with small molecule inhibitors, the researchers demonstrated that the SM09419 drug both reduced XIAP protein levels and synergized with venetoclax, providing a potent combination therapy to “reset” AML tumor sensitivity to venetoclax. In summary, the researchers found that by indirectly inhibiting another anti-apoptotic protein, XIAP, drug-resistant AML became re-sensitized to venetoclax, the inhibitor of the BCL2 anti-apoptotic protein. The restriction of two anti-apoptotic proteins will force the cancer to find another resistance mechanism or remain susceptible to this combination approach.

“Our work provides potential solutions to cancer therapy resistance,” stated Dr. Jose Mario Pineda, second author and postdoctoral research fellow in the Bradley Lab. From these studies “we ascertain potential combination strategies. Specifically, we identified that loss of splicing factors enhanced the sensitivity of these [AML] cells to venetoclax” and determined that by inhibiting these splicing factors, there was a reduction of XIAP anti-apoptotic protein. “Lastly, we found that our novel compound SM09419 [that inhibits splicing factors and causes mis-splicing of XIAP anti-apoptotic protein] also overcomes clinical resistance to venetoclax in patient-derived xenograft models of AML. Our results demonstrate the important role of splicing factors in regulating AML response to BCL2 inhibition,” concluded Dr. Pineda. “We hope our study spurs the production of novel compounds that target splicing factors and the investigation of these drugs to improve AML patient outcomes in response to venetoclax treatment.”

The spotlighted research was funded by the Edward P. Evans Foundation, the National Institutes of Health, the Leukemia & Lymphoma Society, the Mark Foundation for Cancer Research, the Paul G. Allen Frontiers Group, the NCI Cancer Center Support Grant, the Cycle for Survival, and the Marie-Josée and Henry R. Kravis Center for Molecular Oncology.

Fred Hutch/University of Washington/Seattle Children's Cancer Consortium member Robert Bradley contributed to this work.

Wang E, Pineda JMB, Kim WJ, Chen S, Bourcier J, Stahl M, Hogg SJ, Bewersdorf JP, Han C, Singer ME, Cui D, Erickson CE, Tittley SM, Penson AV, Knorr K, Stanley RF, Rahman J, Krishnamoorthy G, Fagin JA, Creger E, McMillan E, Mak CC, Jarvis M, Bossard C, Beaupre DM, Bradley RK, Abdel-Wahab O. 2022. Modulation of RNA splicing enhances response to BCL2 inhibition in leukemia. Cancer Cell. S1535-6108(22)00588-8.