Post doctoral fellow Emory University Atlanta, Georgia, United States
Introduction: Single agent venetoclax (Ven) is effective only in a minority of MM, i.e. 40% of t(11;14)-MM, underscoring the need to better understand the basis of Ven efficacy. We previously reported that low electron transport chain (ETC) activity and reduced oxidative phosphorylation (OXPHOS) are predictive biomarkers of sensitivity to the BCL-2 antagonist, Ven in MM. Moreover, ETC blockade sensitized Ven-resistant MM. Given that heme plays a central role in regulating ETC activity, facilitating catalytic and electron transfer reactions, and regulating assembly of the respiratory complexes, we investigated the role of heme in regulating MM Ven sensitivity.
Methods: Heme (iron protoporphyrin IX) quantification was performed using a fluorometric assay designed to measure the fluorescence of protoporphyrin IX upon the release of iron from heme. We employed a glass-based antibody array for broad-scope protein phosphorylation profiling of MM cell lines treated with hemin (oxidized heme). MM cell lines treated with heme biosynthesis, MEK/ERK and a panel of kinase and metabolic inhibitors were evaluated for viability by flow cytometry.
Results: We recently showed that a more B-cell-like program was associated with Ven sensitivity, thus using the more B-cell like CD2 gene expression subtype for Ven sensitivity we compared the CD1 vs CD2 gene expression subgroups of MM from the CoMMpass trial. This analysis found the CD2 subgroup to exhibit reduced heme biosynthesis gene expression while exhibiting elevated levels of the heme exporters FLVCR1b and ABCG2. Correspondingly, we found Ven sensitive (VS) t(11;14) MM to exhibit i) reduced intracellular heme; ii) reduced rate-limiting ALAS1 enzyme expression and lastly; iii) reduced uptake of 5-aminolevulinic acid (5-ALA), the first committed heme biosynthesis precursor; in line with our previous reports showing VS MM exhibits reduced ETC activity. Supportive of reduced heme in t(11;14) correlating with Ven sensitivity, we observed hemin and protoporphyrin IX supplementation to reverse Ven sensitivity. Suppression of heme synthesis by inhibiting ALAS1 (the rate-limiting step in heme biosynthesis) or ferrochetalase (FECH, the last step in heme biosynthesis), with a newly developed FECH inhibitor promoted Ven sensitivity both in Ven-resistant MM cell lines and primary MM patient samples. Kinase profiling and pharmacological interrogation revealed the RAS-RAF-MEK axis and MCL-1 induction to regulate hemin-induced Ven resistance.
Conclusions: Our results mechanistically illustrate a role for heme in modulating proximity to the apoptotic threshold with broader translational implications. Heme can be sourced from both extrinsic sources such as through diet and cells in the extrinsic milieu or through de novo synthesis, underscoring the importance of investigating heme metabolism in MM therapy.
