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Truncating mutations in the terminal exon of protein phosphatase Mg2+/Mn2+ 1D (PPM1D) have been identified in clonal hematopoiesis and myeloid neoplasms, with a striking enrichment in patients previously exposed to chemotherapy. In this study, we demonstrate that truncating PPM1D mutations confer a chemoresistance phenotype, resulting in the selective expansion of PPM1D-mutant hematopoietic cells in the presence of chemotherapy in vitro and in vivo. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease mutational profiling of PPM1D in the presence of chemotherapy selected for the same exon 6 mutations identified in patient samples. These exon 6 mutations encode for a truncated protein that displays elevated expression and activity due to loss of a C-terminal degradation domain. Global phosphoproteomic profiling revealed altered phosphorylation of target proteins in the presence of the mutation, highlighting multiple pathways including the DNA damage response (DDR). In the presence of chemotherapy, PPM1D-mutant cells have an abrogated DDR resulting in altered cell cycle progression, decreased apoptosis, and reduced mitochondrial priming. We demonstrate that treatment with an allosteric, small molecule inhibitor of PPM1D reverts the phosphoproteomic, DDR, apoptotic, and mitochondrial priming changes observed in PPM1D-mutant cells. Finally, we show that the inhibitor preferentially kills PPM1D-mutant cells, sensitizes the cells to chemotherapy, and reverses the chemoresistance phenotype. These results provide an explanation for the enrichment of truncating PPM1D mutations in the blood of patients exposed to chemotherapy and in therapy-related myeloid neoplasms, and demonstrate that PPM1D can be a targeted in the prevention of clonal expansion of PPM1D-mutant cells and the treatment of PPM1D-mutant disease.

Lenalidomide, an immunomodulatory drug (IMiD), is highly active and broadly used for the treatment of multiple myeloma. Despite high initial remission rates, patients frequently relapse and become resistant to the drug. Comprehensive analyses of gene mutations and RNA expression have identified inactivating mutations and RNA downregulation in cereblon (CRBN), the primary target of lenalidomide, in some of the resistant patients. However, the underlying resistance mechanism for the majority of cases remains unknown. Here, we performed quantitative tandem mass tag (TMT)-based proteomic analyses and RNA sequencing in five paired pre-treatment and relapse samples from multiple myeloma patients treated with drug combinations comprising lenalidomide to identify changes in protein expression associated with resistance. Using a stringent cut-off with an adjusted P value < 0.1 and log2 fold change (FC) > 2, we found 7 proteins to be significantly upregulated and 10 proteins to be downregulated in the relapsed versus pre-treatment multiple myeloma samples. Of these 17 deregulated proteins at relapse, only two were also found to be deregulated on the RNA expression level (adjusted P value < 0.1) as assessed by RNA sequencing. In general, correlation between protein expression levels and RNA expression levels were weak (median Pearson correlation coefficient r=0.35). Among the top upregulated proteins in relapse samples was cyclin-dependent kinase 6 (CDK6) with an average log2 FC of 2.1. Protein and RNA levels of CDK6 showed only weak correlation (r=0.4) and CDK6 RNA was not differentially expressed between the relapse and pretreatment samples. To validate the findings of the proteomic analysis, we assessed CDK6 protein levels by western blot in additional patient samples obtained at diagnosis (N=4) and at relapse (N=9). This confirmed a high CDK6 protein expression in 6 of 9 relapse samples while CDK6 could not be detected in the 4 pre-treatment samples. In order to determine the impact of CDK6 on drug sensitivity, we overexpressed CDK6 using either a retro- or lentiviral vector system in multiple myeloma cell lines. In two multiple myeloma cell lines tested, MM.1S and OPM2, CDK6 overexpression reduced sensitivity to lenalidomide and pomalidomide, but not to melphalan, bortezomib, or dexamethasone. To examine whether lowered IMiD-sensitivity can be overcome by CDK6 inhibition, we treated multiple myeloma cell lines either with the CDK6 inhibitor palbociclib, an IMiD-based CDK6-selective proteolysis targeting chimera (PROTAC) or a non-selective CDK6-PROTAC which is also capable of pomalidomide-mediated degradation of IKZF1 and IKZF3 (Brand et al., Cell Chem Biol 2019). Both palbociclib and CDK6-selective PROTAC as single treatments had only mild effects on the majority of multiple myeloma cells, implying that multiple myeloma cells are generally not dependent on CDK6. In contrast, the combination treatment of palbociclib with lenalidomide, or the non-specific CDK6/IKZF1/IKZF3-targeting PROTAC significantly inhibited proliferation, producing synergistic effects on the decrease of myeloma cell viability in 6 multiple myeloma cell lines, including those with a low IMiD sensitivity like RPMI-8226 and L363. This demonstrates that CDK6 inhibition or degradation enhances the cytotoxic effects of IMiDs. In order to investigate a potential mechanism for the synergistic effects of CDK6 inhibition and IMiDs, we analyzed protein levels in treated cells. CDK6 inhibition or degradation had no effect on CRBN protein levels nor on lenalidomide-induced degradation of IKZF1 and IKZF3. In contrast, combined degradation of CDK6, IKZF1, and IKZF3 revealed decreased protein levels of c-MYC, which was not observable in cells treated with palbocicilib, CDK6-selective PROTAC or pomalidomide alone. In conclusion, quantitative proteomics in primary multiple myeloma samples identified new druggable candidates including CDK6 in relapse that were overlooked by RNA expression analyses. Inhibition of CDK6 by palbociclib or a PROTAC sensitizes multiple myeloma cells to IMiDs and results in synergism when used in combination. Disclosures Bohl: Pfizer: Honoraria. Bullinger:Menarini: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; Seattle Genetics: Honoraria; Janssen: Honoraria; Jazz Pharmaceuticals: Honoraria; Amgen: Honoraria; Astellas: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Daiichi Sankyo: Honoraria; Gilead: Honoraria; Hexal: Honoraria; Bayer: Other: Financing of scientific research; Abbvie: Honoraria. Kroenke:Celgene: Consultancy, Honoraria; Takeda: Consultancy.

One of the adverse consequences of chemotherapy exposure is the development of therapy-related myeloid neoplasms (t-MNs). However, the cause and origin of most t-MNs are unknown, and the prognosis remains dismal. Novel work has shown that in addition to TP53, PPM1D is selectively mutated in 15% of therapy related MDS (Lindsley et al., ASH Abstract). Truncating mutations of PPM1D are also found to commonly occur in clonal hematopoiesis (Jaiswal et al., NEJM 2014; Genovese et al., NEJM 2014; Xie et al., Nat Med 2014), as well as in the blood of cancer patients, particularly after exposure to chemotherapy (Ruark et al., Nature 2013; Swisher et al., JAMA Oncol 2016). We hypothesized that PPM1D truncating mutations confer chemotherapy resistance, causing selective outgrowth of PPM1D mutant hematopoietic stem cells in states of genotoxic stress. In addition, since PPM1D mutations lead to a gain of function, we examined the potential of targeting PPM1D-mutant cells pharmacologically. The protein phosphatase PPM1Dis a direct regulator of TP53 activity and the DNA damage response pathway (Fiscella et al., PNAS 1997). Consequently, gain-of-function PPM1D mutations lead to decreased TP53 activity. To examine whether PPM1D mutations drive chemotherapy resistance through an abrogation of the TP53 dependent DNA damage response, we engineered PPM1D-mutant subclones using the CRISPR-Cas9 system in the TP53 wild-type AML cell line MOLM-13. PPM1D exon 6 truncation led to increased expression of PPM1D and resistance to DNA damaging agents, including cytarabine, cyclophosphamide and cisplatin. In addition, PPM1D-mutant cells exhibited a selective advantage over wild-type cells in the presence of chemotherapy, expanding 100-fold over a 24-day period (Fig. 1). While treatment with chemotherapy induced phosphorylation of Chk1 and p53, cell cycle arrest and apoptosis in wild-type cells, this response was abrogated in PPM1D-mutant cells due to gain of function of PPM1D. Using phosphoproteomic analysis, we further demonstrate decreased phosphorylation of known and novel targets in PPM1D-mutant compared to wild-type cells. We next investigated the effect of PPM1D mutation on normal marrow progenitors in response to chemotherapy treatment in vivo. We performed a competition experiment in which mouse bone marrow c-Kit+ cells expressing Cas9 were transduced with guide RNAs targeting exon 6 of PPM1D or a control guide, and then transplanted into mice in a 1:5 ratio. We observed a selective outgrowth of PPM1D-mutant myeloid cells in the peripheral blood of mice after exposure to chemotherapy. In addition, we found expansion of PPM1D-mutant cells in the lineage negative, c-Kit+ Sca-1+ population, which is enriched for hematopoietic stem cells and multipotent progenitors, indicating that PPM1D-mutant stem and progenitor cells have a competitive advantage over wild-type cells after exposure to genotoxic stress. The generation of a selective, allosteric inhibitor of PPM1D (Gilmartin et al., Nat Chem Biol 2014) allowed us to examine whether pharmacologic inhibition of PPM1D decreases the chemotherapy resistance or survival of PPM1D-mutant cells. We found that PPM1D-mutant cells have a significantly increased sensitivity to PPM1D inhibition when compared to wild-type controls. In addition, PPM1D inhibitor treatment was able to re-sensitize mutant cells to chemotherapy and abrogate the selective outgrowth of PPM1D-mutant cells during chemotherapy exposure. Lastly, we demonstrate that the proteome-wide phosphorylation profile characteristic of PPM1D-mutant cells can be reversed through treatment with the PPM1D inhibitor. In sum, these results demonstrate that PPM1D mutations confer a competitive advantage to hematopoietic stem cells undergoing genotoxic stress by abrogating the DNA damage response, and are likely to be the initiating mutation in a large proportion of t-MNs. Due to the gain-of-function nature of this mutation, PPM1D-mutant cells are differentially sensitive to treatment with a PPM1D inhibitor. PPM1D inhibition may therefore provide an opportunity for the prevention and targeted treatment of hematologic malignancies that harbor PPM1D mutations. Figure 1 PPM1D mutant cells have a competitive advantage under the selective pressure of chemotherapy (cytarabine) treatment. Figure 1. PPM1D mutant cells have a competitive advantage under the selective pressure of chemotherapy (cytarabine) treatment. Disclosures No relevant conflicts of interest to declare.