Deletion (del) of 17p involving the p53 tumor suppressor (TP53) remains an adverse prognostic factor in multiple myeloma (MM) despite the use of novel agents as well as high-dose chemotherapy with autologous stem cell rescue. Genomic TP53 deletion can cause haploinsufficiency of nearby genes, such as RNA polymerase II subunit A (POLR2A), which ia also located on 17p13.1. We therefore hypothesized that del 17p could reduce POLR2A expression and enhance sensitivity to a-Amanitin, a potent and specific inhibitor of POLR2A and RNA polymerase III.
Pre-clinical studies were performed using HDP101, a monoclonal antibody-drug conjugate (ADC) targeting BCMA linked to a-Amanitin, along with unconjugated BCMA antibody, a-Amanitin, and a non-targeting control ADC in myeloma cell line models. The latter included H929, MM1.S, and MOLP-8 TP53 wild-type (WT) lines and isogenic cells in which TP53 had been knocked out (KO) using CRISPR/Cas9 genome editing techniques. To further model del 17p and POLR2A haploinsufficiency, POLR2A expression was knocked down using shRNAs.
Analysis of the Multiple Myeloma Research Foundation CoMMpassSM database revealed that del 17p13 by SeqFISH was associated with a significant reduction in POLR2A expression by RNASeq (26.05431 fragments per kilobase of transcript per million mapped reads (FPKM) with WT vs. 19.2983 FPKM with del 17p13; p<0.0001). Also, patients within the lower quartile of POLR2A expression, which included those with and without del 17p, had an inferior overall survival (p<0.0011) and a trend towards a worse progression-free survival, suggesting that low POLR2A levels by themselves are an adverse feature. The POLR2A inhibiting anti-BCMA/a-Amanitin conjugate HDP101 induced a time- and dose-dependent reduction in myeloma cell viability post 96-hours of drug exposure starting at concentrations as low as in the picomolar range. This reduction with HDP101 was much greater than that seen with controls, which included a-Amanitin alone, the anti-BCMA antibody without a-Amanitin, or an anti-digoxigenin antibody conjugated to a-Amanitin. When myeloma cells were co-cultured with HS-5 human marrow stromal cells, only a-Amanitin and, to a much greater extent, HDP101 induced a loss of cell viability in myeloma cells, while the stromal cells were spared by HDP101. Loss of cell viability due to HDP101 was associated with induction of apoptosis as judged by the appearance of an increased population of cells that had a sub-G0/G1 DNA content, and that stained positively with Annexin V. Moreover, HDP101 treatment caused the appearance of cleaved fragments of Caspase 9 and 3, and loss of the mitochondrial trans-membrane potential. H929, MM1.S, and MOLP-8 TP53 KO cells were more sensitive to both HDP101 and, less potently to a-Amanitin than were their isogenic TP53 WT parental cells. As H929 cells expressed high levels of BCMA regardless of TP53 or POLR2A status, these were further examined for their sensitivity to HDP101. Notably, the preferential impact upon TP53 KO cells was associated with increased expression of Activating transcription factors -4 and -6, suggesting enhanced induction of endoplasmic reticulum stress, and at least two arms of the unfolded protein response. Interestingly, shRNA-mediated knockdown (KD) of POLR2A expression alone was sufficient to increase baseline levels of apoptosis in both H929 TP53 WT and KO cells, with a greater impact in the latter, supporting the promise of this target. Importantly, KD of POLR2A expression to further model del 17p was also associated with enhanced sensitivity to HDP101 in both the TP53 WT and KO cells compared to the control treatments. Evaluation of HDP101 in primary samples and with in vivo models is underway, and will be presented at the Annual Meeting. These studies were supported by a Leukemia & Lymphoma Society Specialized Center of Research (SCOR-12206-17).
Our preliminary data support the possibility that del 17p myeloma may have a therapeutic vulnerability to the POLR2A inhibitor a-Amanitin through loss of TP53, and that this sensitivity is further enhanced by decreased POLR2A expression, which is common among del 17p patients. Moreover, they suggest that HDP101 is a novel potent and specific therapeutic that could show enhanced activity in the clinic especially against high-risk multiple myeloma, where effective therapies are still needed to improve patient outcomes.
Pahl:Heidelberg Pharma AG: Employment. Orlowski:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; Genentech: Consultancy; Poseida: Research Funding; BioTheryX, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millenium Pharmaceuticals: Consultancy, Research Funding.
Asterisk with author names denotes non-ASH members.