Mitochondrial Apoptotic Priming Measured by BH3 Profiling Regulates Clinical Response to Chemotherapy in Myeloma and Acute Lymphoblastic Leukemia and Explains Therapeutic Index
Authors: Chonghaile Triona Ni , Sarosiek Kristopher , Ryan Jeremy , Moore Vicki Del Gaizo , Tai Yu-Tzu , Richardson Paul G. , Anderson Kenneth C. , Stone Richard M. , DeAngelo Daniel J , Mitsiades Constantine S. , Sallan Stephen E. , Hirsch Michelle , Silverman Lewis B. , Carrasco Ruben , Letai Anthony G. 
Published: October 2019

Abstract
Abstract 1442
Cytotoxic chemotherapy, still a mainstay of current cancer treatment, targets ubiquitous elements such as DNA and microtubules. Despite decades of clinical use of chemotherapy, determinants of response to such treatments are poorly understood. Here, we showed that clinical response to cytotoxic agents is largely regulated by the initial proximity of tumors' mitochondria to the apoptotic threshold. Cells that are close to the apoptotic threshold we refer to as “primed” for death. Priming is assessed by a functional assay we call BH3 profiling that measures mitochondrial response to standardized death signals in the form of peptides from the pro-apoptotic BH3-only proteins. We assessed priming across a total of 51 patient samples, 25 of which had clinical follow-up. Priming correlated to % M-protein reduction, a marker of reduced disease burden in multiple myeloma following treatment (spearman r = 0.8039, P= 0.00005 Fig 1A). Patient's with highly primed mitochondria also demonstrated a better clinical response in both multiple myeloma (P= 0.001) and in acute lymphoblastic leukemia (P= 0.0119). Pediatric ALL responds better to therapy and has a much higher long term survival rate than adult ALL (80–90% vs. 40%) (Pui et. al N Engl J Med, 2006). We found that pediatric ALL samples were more primed than adult (P = 0.007). To demonstrate the relationship between priming and chemosensitivity we perturbed priming by co-culture with stroma or by pretreatment with ABT-737 and demonstrated that altering priming changed chemosensitivity predictably.
By far the most important dichotomy in sensitivity to chemotherapy is between normal tissues and cancer cells. Differences in proliferation rate, the classic explanation for this dichotomy offers only modest explanatory power with many exceptions. We therefore tested whether differential mitochondrial apoptotic priming was an important determinant of the therapeutic index from cytotoxic chemotherapy. We directly tested the mitochondrial priming of a series of normal tissues from both human (8 tissues) and mouse (8 tissues). Significantly, we found that most normal tissues along with chemoresistant (Serous borderline ovarian, Renal and Endometrial) cancers had relatively low priming (Fig 1B). Conversely chemosensitive cancers and normal hematopoietic tissues were highly primed. Differential mitochondrial priming may provide a novel explanation for the otherwise puzzling existence of a therapeutic window for cytotoxic agents. This newly appreciated determinant of response to chemotherapy could be exploited to rationally improve efficacy of cytotoxic agents or to devise better combinations of drugs.Fig 1:Mitochondrial priming regulates clinical response to chemotherapy in multiple myeloma and acute lyphoblastic leukemia (A) Loss of δΨm caused by the BMF peptide in myeloma patient samples correlates to %M-protein reduction. (B) Comparison of priming among all primary human cancers and normal tissues. The cancers with clinical follow up were classified as known chemosensitive or known chemoresistant. Cancers classified as typically chemoresistant (Serous borderline n=3, Endometrial n=3 and Renal n=3) or typically chemosensitive (childhood ALL n=17) lacked individual response data. Data shown are mean ± s.d. across all specimens tested. ANOVA was used to demonstrate statistical significance between the different categories with a Tukey's multiple comparison post test. ns - p value > 0.05 and *** p-value <0.001.Fig 1:. Mitochondrial priming regulates clinical response to chemotherapy in multiple myeloma and acute lyphoblastic leukemia (A) Loss of δΨm caused by the BMF peptide in myeloma patient samples correlates to %M-protein reduction. (B) Comparison of priming among all primary human cancers and normal tissues. The cancers with clinical follow up were classified as known chemosensitive or known chemoresistant. Cancers classified as typically chemoresistant (Serous borderline n=3, Endometrial n=3 and Renal n=3) or typically chemosensitive (childhood ALL n=17) lacked individual response data. Data shown are mean ± s.d. across all specimens tested. ANOVA was used to demonstrate statistical significance between the different categories with a Tukey's multiple comparison post test. ns - p value > 0.05 and *** p-value <0.001.

Disclosures:
Richardson: Millennium:; Celgene:; Johnson & Johnson:; Novartis:; Bristol Myers Squibb:. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees. Mitsiades:Millennium: Consultancy, Honoraria. Sallan:Enzon Pharmaceuticals: Honoraria. Letai:Eutropics Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees.