Engineering dual-specificity PPM1D inhibitors to enhance therapeutic response and overcome chemoresistance in TP53-mutant malignancies Free

Background and Objective: Cytotoxic chemotherapy remains a foundational treatment for many cancers, yet its effectiveness is often constrained by intrinsic or acquired resistance and dose-limiting toxicities. Strategies that enhance the therapeutic index of chemotherapy could benefit a broad spectrum of cancer patients, most notably those with hematologic malignancies. The serine/threonine phosphatase PPM1D (WIP1) is a negative regulator of the DNA damage response (DDR) and p53 activation, and PPM1D is recurrently activated in myeloid malignancies through c-terminal truncating mutations. We and others have demonstrated that pharmacological inhibition or genetic loss of PPM1D sensitizes TP53-wild-type leukemia cells to DNA-damaging agents. However, whether the therapeutic utility of PPM1D inhibition extends beyond TP53 status and DNA damage pathways remains an unanswered biological and therapeutic question. Here, we report the results of a chemicogenomic study to identify and characterize novel, dual-specificity compounds that enhance the activity of chemotherapy through PPM1D- and TP53-dependent and independent mechanisms. Our findings provide the biological and therapeutic framework for developing classes of compounds to enhance treatment response in both TP53-wildtype and -mutant malignancies.

Methods: We screened a panel of acute myeloid leukemia (AML) cell lines to determine whether PPM1D inhibition with the small molecule GSK2830371, or additional compounds that we recently generated, sensitized cells to different classes of chemotherapy. We also performed multiplexed screens of more than 700 cancer cell lines using daunorubicin with or without GSK2830371, as well as over 400 cancer cell lines using vincristine with or without GSK2830371. Follow-up studies in isogenic cell line models evaluated the sensitivity to additional chemotherapeutics and targeted agents. Structure-activity relationship (SAR) analyses were performed using more than 50 PPM1D inhibitor analogs with defined biochemical properties. The roles of PPM1D, TP53, and ABCB1 (MDR1) were evaluated using CRISPR knockout, immunoblotting, drug accumulation assays, antibody binding assays, and ATPase activity profiling.

Results: GSK2830371 sensitized TP53-wild-type cells to most chemotherapeutic agents, but, unexpectedly, also sensitized TP53-mutant cells to daunorubicin. A chemical structure similarity analysis of daunorubicin to known therapeutics highlighted vinca alkaloids and taxanes, which we subsequently tested. Indeed, GSK2830371 also sensitized TP53-mutant cells to these chemotherapy classes (which we further confirmed using TP53 isogenic cell lines), but not to other cell cycle modulators, such as CDK1 or Eg5 inhibitors, which suggests that the effect of GSK2830371 was not due to enhanced on-target activity. Moreover, we used PPM1D isogenic cell lines and compounds that we previously generated with differing PPM1D binding to show that the chemosensitization effect was independent of PPM1D protein levels and function. We then used a multiplexed screening platform of more than 400 cell lines and confirmed that GSK2830371 sensitized both TP53-wild-type and -mutant cells to daunorubicin and vincristine. Moreover, we performed biomarker association analyses using data from these hundreds of cell lines and found that the sensitization activity of GSK2830371 was significantly linked to the expression of the drug efflux pump, MDR1 (encoded by ABCB1). Using MDR1 isogenic cell lines, we confirmed that the chemosensitization phenotype of GSK2830371 and the other PPM1D inhibitors was indeed MDR1-dependent. We found that GSK2830371 and select analogs increased intracellular accumulation of daunorubicin, enhanced the binding of antibodies that recognize the active conformation of MDR1, and modified the ATPase activity and efflux kinetics of MDR1. Finally, we performed extensive structure-activity relationship analyses to identify the sulfonamide moiety in our compounds as the critical regulator of MDR1, but not PPM1D, engagement.

Conclusions: Our data highlight new biological and medicinal chemistry strategies to engineer dual specificity of PPM1D inhibitors, enhancing chemotherapeutic activity via PPM1D- and TP53-dependent and -independent mechanisms.