Molecular Consequences and Targeting of PPM1D Mutations and Overexpression in Myeloproliferative Neoplasm Stem Cells
Authors: Clementelli Cara , Mia MD Babu , Haines Sarah , Schaniel Christoph , Olszewska Malgorzata , Arandela Joann , Elghaity-beckley Sebastian , Klingborg Sophie , Hoque Zinedine , Lu Min , Papapetrou Eirini , Hoffman Ronald , Marcellino Bridget Kelly 
Published: December 2024

Truncating mutations of Protein Phosphatase Mg2+/Mn2+ Dependent 1D (PPM1D) found in myeloproliferative neoplasms (MPNs) and other myeloid malignancies cause loss of the C-terminal degradation domain and result in increased protein levels of PPM1D (Grinfeld et al. 2018., Hsu et al. 2018). We have previously shown that myelofibrosis (MF) patients with unmutated (wild-type, WT) PPM1D overexpress PPM1D transcript in their hematopoietic stem/progenitor cells (HSPCs) (Marcellino et al., ASH 2022).

To test if PPM1D overexpression occurs in MPN more broadly, we measured PPM1D transcript levels in a large cohort of patients (n=82) of all MPN subtypes (Polycythemia Vera, Essential Thrombocythemia, MF, MPN-blast phase). We found PPM1D overexpressed in HSPCs of all subtypes (fold change average 83.9 vs normal donor, p=0.0002). PPM1D expression was higher in JAK2V617F+ compared to CALR-mutated, samples (43.4 vs 13.4 fold change, respectively, p=0.01). We also found PPM1D increased at the protein level in primary MF samples.

To elucidate the impact of PPM1D mutation and overexpression on the MPN clone, we reprogrammed mononuclear cells from a JAK2V617F+, PPM1D c.1405A&gt;T MPN patient into induced pluripotent stem cell (iPSC) lines, generating both a JAK2V617F+, PPM1D wild-type (WT) line and a JAK2V617F+, PPM1D mutant (MUT) line, thus obtaining isogenic lines with and without the PPM1D mutation. We confirmed increased PPM1D protein expression in PPM1D-MUT compared to the PPM1D-WT iPSC derived HSPCs (p=0.01).

To understand the downstream effects of PPM1D mutation and overexpression, we performed single-cell RNA sequencing of CD34+ cells differentiated from PPM1D-MUT and PPM1D-WT MPN iPSC lines, which revealed significant alterations in critical regulatory pathways. PPM1D-MUT iPSC HSPCs exhibited downregulation of the p53 pathway, corroborated by western blot analysis of phosphorylated p53 protein. Notably, key DNA repair genes BRCA1, BRCA2, CHEK1, and CHEK2 were significantly downregulated, indicating compromised DNA damage response (Bartek et al., 2003) with CHEK2 protein downregulation further validated by western blot. Concurrently, we observed upregulation of the cell cycle promoters CDK2, CDK6 and CDC25B, coupled with downregulation of CDKN1A, consistent with enhanced proliferative capacity (Malumbres et al., 2009). Analysis of apoptosis regulators revealed upregulation of anti-apoptotic BCL2 and downregulation of pro-apoptotic BBC3 and TP53BP2, suggesting a shift towards cell survival (Cory et al., 2002).

We then assessed the effects of targeting PPM1D in the PPM1D-MUT and WT iPSC-HSPCs utilizing GSK2830371, an allosteric inhibitor of PPM1D (PPM1Di). Treatment of the PPM1D-MUT HSPCs with the PPM1Di (1µM or 3 µM) resulted in a significant reduction in colony formation (1 uM, p=0.003; 3 uM p =0.001) whereas the PPM1D-WT HSPCs were not affected (1 µM, p=0.06; 3 µM p =0.11). These findings were corroborated with flow cytometric studies showing greater apoptosis of PPM1D-MUT than PPM1D-WT HSPCs with treatment.

PPM1D directly interacts with HDM2, another key negative regulator of P53. Clinical trials have revealed promise in targeting HDM2 in MPN patients (Mascarenhas et al., 2019). Additionally, we have previously shown effectiveness of combinatorial PPM1D and HDM2 inhibition in primary MF HSPCs without PPM1D mutation in vitro (Marcellino et al., ASH 2022). We thus tested this combination in PPM1D-MUT and WT iPSC HSPCs. Addition of low dose HDM2i (50 nM) to PPM1Di (1 or 3 µM) significantly reduced colony formation compared to PPM1Di alone (PPM1D-MUT HSPCs 1µM p=0.01, 3 µM p=0.01; PPM1D-WT 1µM p=0.16, 3 µM p=0.01). We then performed synergistic studies to evaluate the PPM1Di and HDM2i combination in a primary MF sample utilizing HDM2i at doses 0-200 nM and PPM1Di at doses 0-10 µM. Drug synergy was demonstrated at multiple time points of treatment with significant cooperative activity occurring with PPM1Di doses as low as 0.5 µM and HDM2i at 25 nM (day 6 ZIP synergy score 26.97, p=3.65e-09).

These findings collectively delineate a multifaceted mechanism by which PPM1D mutations confer a selective advantage to MPN HSPCs and provide a strong rationale for targeting PPM1D to deplete MPN HSPCs. We also demonstrate that MPN iPSCs and MPN primary specimens are complementary systems to enable the elucidation of the biology, identification of targets and evaluation of drug susceptibility of MPN HSPCs.