Combined inhibition of CTPS1 and ATR is a metabolic vulnerability in p53‐deficient myeloma cells

In multiple myeloma, as in B-cell malignancies, mono- and especially bi-allelic <i>TP53</i> gene inactivation is a high-risk factor for treatment resistance, and there are currently no therapies specifically targeting p53 deficiency. In this study, we evaluated if the loss of cell cycle control in p53-deficient myeloma cells would confer a metabolically actionable vulnerability. We show that CTP synthase 1 (<i>CTPS1</i>), which encodes a CTP synthesis rate-limiting enzyme essential for DNA and RNA synthesis in lymphoid cells, is overexpressed in samples from myeloma patients displaying a high proliferation rate (high <i>MKI67</i> expression) or a low p53 score (synonymous with <i>TP53</i> deletion and/or mutation). This overexpression of <i>CTPS1</i> was associated with reduced survival in two cohorts. Using scRNA-seq analysis in 24 patient samples, we further demonstrate that myeloma cells in the S or G2/M phase display high <i>CTPS1</i> expression. Pharmacological inhibition of CTPS1 by STP-B induced cell cycle arrest in early S phase in isogenic NCI-H929 or XG7 <i>TP53</i> ^+/+, <i>TP53</i> ^-/-, and <i>TP53</i> ^R175H/R175H cells and in a <i>TP53</i> ^-/R123STOP patient sample. The functional annotation of transcriptional changes in 10 STP-B-treated myeloma cell lines revealed a decrease in protein translation and confirmed the blockade of cells into the S phase. The pharmacological inhibition of ATR, which governs the intrinsic S/G2 checkpoint, in STP-B-induced S-phase arrested cells synergistically induced cell death in <i>TP53</i> ^+/+, <i>TP53</i> ^-/-, and <i>TP53</i> ^R175H/R175H isogenic cell lines (Bliss score >15). This combination induced replicative stress and caspase-mediated cell death and was highly effective in resistant/refractory patient samples with <i>TP53</i> deletion and/or mutation and in <i>TP53</i> ^-/- NCI-H929 xenografted NOD-scid IL2Rgamma mice. Our in vitro, ex vivo, and in vivo data provide the rationale for combined CTPS1 and ATR inhibition for the treatment of p53-deficient patients.