CTPS1 Is a Novel Therapeutic Target in Multiple Myeloma That Synergizes with Inhibition of ATR, CHEK1 or WEE1

Introduction Recent years have seen advances in multiple myeloma (MM) therapy, with favorable risk patients achieving overall survival (OS) of 10 years or more. For a subset of patients, however, outcomes remain poor with median OS less than 2 years, highlighting the need for new treatments. Inhibition of DNA synthesis is an effective mechanism to kill tumor cells that is exploited by several chemotherapy drugs. Precision targeting of these pathways has the potential to deliver improved efficacy and tolerability; however, attempts to date have been limited by the essential nature of DNA synthesis to all dividing cells. Conversion of UTP to CTP, the rate limiting step in pyrimidine synthesis, is unusual in being catalyzed by two homologous enzymes, CTP synthase 1 (CTPS1) and CTPS2. Of note, human genetic studies have identified an essential and non-redundant role for CTPS1 in lymphoid cell proliferation whereas CTPS2 is able to compensate outside the hematopoietic system, raising the possibility that selective inhibition of CTPS1 could deliver anti-tumor efficacy without systemic toxicity.

Methods Gene expression was interrogated in publicly available RNA-seq datasets of patient derived MM samples at different stages of disease progression. CTPS1 knock-out (KO) cell lines were generated using a two vector CRISPR/Cas9 system. The impact of the highly-specific CTPS1 inhibitor STP938 (Step Pharma) on cell proliferation was tested in 12 MM cell lines using a WST-8 based assay. Cell cycle and apoptosis were analyzed by flow cytometry staining for propidium iodide or annexin V/7AAD, respectively. DNA damage response (DDR) pathway activation was assessed by immune blotting for phosphorylated CHEK1 and CHEK2. Double stranded DNA (dsDNA) breaks were assessed by flow cytometric analysis of phosphorylated H2AX (γH2AX). Synergy was analyzed in vitro in a drug response matrix with ZIP scores calculated by Synergyfinder 2.0.

ResultsIn silico analysis revealed a strong upregulation of genes involved in pyrimidine biosynthesis during MM disease progression (GSE13591). Specifically, CTPS1 expression showed a stepwise increase from normal plasma cells through monoclonal gammopathy of uncertain significance (MGUS) to MM, with the highest expression observed in plasma cell leukemia (PCL) (Figure A), findings that were confirmed in an independent dataset (GSE6477). Additionally, CTPS1 expression was found to be upregulated in patients with a previously described proliferative gene expression signature known to be associated with poor outcomes (GSE19784, GSE2658); consistent with this, high CTPS1 expression was associated with reduced overall survival in MM patients (GSE2658, GSE9782).

Knock-out of CTPS1 in RPMI8226 and SK-MM-1 cell lines resulted in growth inhibition and apoptosis, confirming the essential nature of this pathway. Exposure of MM cell lines with the CTPS1 inhibitor STP938 (>1,300-fold selectively over CTPS2), resulted in potent inhibition of cell viability for 6 of 12 MM cell lines tested (IC₅₀ sensitive lines 33 - 234 nM, resistant lines >5 µM). STP938 induced S phase arrest and time- and concentration-dependent apoptosis in sensitive cell lines. This was associated with DDR pathway activation, as evidenced by phosphorylation of CHEK1 and CHEK2, and accumulation of dsDNA breaks in early S phase, as assessed by combined cell cycle and γH2AX analysis. Of note, S phase arrest, DDR activation and dsDNA breakage was also observed in cell lines that were resistant to STP938. Combining CTPS1 inhibition by STP938 with inhibitors of different components of the DDR pathway demonstrated strong synergy, with consistent results observed when combining STP938 with inhibitors of either ATR (ceralasertib, VE-821), CHEK1 (rabusertib, SRA737) or WEE1 (adavosertib) (Figure B). Importantly, these combinations showed synergistic activity against cell lines sensitive and resistant to single agent STP938.

Conclusion These preclinical data identify inhibition of CTPS1 as a potential therapeutic target in the treatment of MM, either alone or in combination with an inhibitor of the DDR pathway. The ability of the homologous CTPS2 enzyme to compensate for CTPS1 loss outside the hematopoietic system suggests that inhibition of CTPS1 will not be associated with significant non-hematological toxicity. Clinical evaluation of STP938 in lymphoid malignancies will start in Q3 this year.

Asnagli:Step Pharma: Current Employment. Schneller:Amgen: Other: Travel Support. Zojer:Janssen: Honoraria; BMS/Celgene: Honoraria; Amgen: Honoraria; Sanofi: Honoraria; Takeda: Honoraria. Schreder:Janssen: Honoraria; AbbVie: Honoraria; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria. Parker:Step Pharma: Current Employment. Bolomsky:Johnson & Johnson: Ended employment in the past 24 months. Beer:Step Pharma: Current Employment.