Selective pharmacologic targeting of CTPS1 shows single-agent activity and synergizes with BCL2 inhibition in aggressive mantle cell lymphoma

Abstract

Innovative therapeutic strategies have emerged over the past decade to improve outcomes for most lymphoma patients. Nevertheless, the aggressive presentation seen in high-risk mantle cell lymphoma (MCL) patients remains an unmet medical need. The highly proliferative cells that characterize these tumors depend on nucleotide synthesis to ensure high DNA replication and RNA synthesis. To take advantage of this vulnerability, STP-B, a clinically available small molecule selectively targeting CTP synthase 1 (CTPS1) has been recently developed. CTPS1 is a key enzyme of the pyrimidine synthesis pathway mediated through its unique ability to provide enough CTP in highly proliferating cells. Herein, we demonstrated that CTPS1 was expressed in all MCL cells, and that its high expression was associated with unfavorable outcomes for patients treated with chemotherapy. Using aggressive MCL models characterized by blastoid morphology, TP53 mutation or polyresistance to targeted therapies, we showed that STP-B was highly effective at nanomolar concentrations in vitro and in vivo, irrespective of these high-risk features. Inhibition of CTPS1 rapidly leads to cell cycle arrest in early S-phase accompanied by inhibition of translation, including of the anti-apoptotic protein MCL1. Consequently, CTPS1 inhibition induced synergistic cell death in combination with the selective BCL2 inhibitor venetoclax, both in vitro and in vivo. Overall, our study identified CTPS1 as a promising target for MCL patients and provided a mechanism-based combination with the BCL2 inhibitor venetoclax for the design of future chemotherapy-free treatment regimens to overcome resistance.

Figure 1.

CTPS1, but not CTPS2, is highly expressed in mantle cell lymphoma and associated with poor prognosis. (A) CTPS1 and CTPS2 expression was assessed by RNA sequencing (RNA-seq) in lymph node (LN) biopsies from 100 mantle cell lymphoma (MCL) patients at diagnosis. (B) CTPS1/2 mRNA levels were analyzed by 3’SRP in MCL cells from bone marrow (BM, N=9) or peripheral blood (PB, N=63) patient samples. (C) CTPS1/2 expression was determined in 11 MCL cell lines (CL) by 3’SRP. Mann-Whitney test was used. **P<0.01, ****P<0.0001. (D) Overall survival probabilities with different CTPS1/2 expression were estimated by the Kaplan-Meier method. Probabilities were calculated on 122 MCL patients treated with rituximab, cyclophosphamide, doxorubicin, prednisone, vincristine (R-CHOP) (public dataset GSE93291). ****P<0.0001. (E) Overall survival probabilities with different CTPS1/2 expression were assessed similarly using data from the LYMA trial (N=98). *P<0.05. NS: not significant; R-DHAP: rituximab, cisplatin, dexamethasone, and high-dose cytarabine; 3’SRP: 3’sequencing-RNA profiling.

Figure 2.

Selective CTPS1 targeting reduces tumor viability in aggressive mantle cell lymphoma preclinical models. (A) Dose response to STP-B was evaluated by CellTiter-Glo (CTG) assay in 11 mantle cell lymphoma (MCL) cell lines treated for 72 hours. (B) Response to STP-B was similarly assessed in JeKo-1 and a derived ibrutinib-resistant JeKo-1 (left graph) and in MAVER-1 and a derived venetoclax-resistant MAVER-1 (right graph). (C) Dose response to STP-B was evaluated by CTG assay in 2 Z138 TP53^KO clones compared to isogenic Z138 TP53^WT cells treated for 72 hours. (D) STP-B efficacy was determined in vivo using Z138 xenograft model. Mice were treated with vehicle (N=5) or 30 mg/kg STP-B (N=5) 4 consecutive days a week for 3 cycles. Tumor size was measured by caliper. (E) STP-B efficacy in vivo was assessed using a disseminated patient-derived xenograft (PDX) model (ibrutinib-resistant, TP53^MUT, blastoid). Left panels: circulating MCL cells count was determined by flow cytometry (human [hu] CD45⁺) 21 days and 28 days after engraftment. Mann-Whitney test was used. **P<0.01. Right panel: survival of PDX mice treated with vehicle (N=5) or STP-B (N=5) was analyzed. Mantel-Cox and Gehan-Breslow-Wilcoxon tests were performed.

Figure 3.

CTPS1 is preferentially expressed in cycling mantle cell lymphoma cells and its inhibition affects cell cycle related transcriptional programs. (A) Nine mantle cell lymphoma (MCL) cell lines were treated with STP-B at half maximal inhibitory concentration (IC₅₀) for 24 hours and genes expression was determined by 3’sequencing-RNA profiling (3’SRP). The top 10 enriched Reactome pathways modulated upon STP-B treatment in MCL cells are depicted. (B) Cell cycle associated genes expression was analyzed upon STP-B treatment. RNA level fold changes (STP-B/untreated) were calculated for genes involved in G1/early-S phases and late-S/G2/M phases. (C) CTPS1/2 expression was compared to MKI67 expression in lymph node (LN) biopsies from MCL patients using either RNA sequencing (RNA-seq) data (N=98, upper panels) or gene expression profiling data (N=122, lower panels). Spearman test was used. (D) CTPS1 and CTPS2 levels were assessed by single-cell RNA-seq in highly proliferating and resting cells from 6 bone marrow (BM) samples from MCL patients (red line: median).

Figure 4.

CTPS1 inhibition results in rapid early S-phase arrest followed by late cell death in mantle cell lymphoma. (A) Cell cycle analysis (bromodeoxyuridine/propidium iodide [BrdU/PI]) was performed in 5 mantle cell lymphoma (MCL) cell lines treated for 24 hours (h) with STP-B at half maximal inhibitory concentration (IC₅₀). Percentage of cells in G1, early-S, late-S and G2/M phases is indicated. Change in cell cycle distribution was calculated. Paired t test was used. *P<0.05, **P<0.01, ****P<0.0001. (B) BrdU/PI analysis was performed in 11 MCL primary samples. Primary cells were co-cultured for 72 hours (h) with L40 cells and cytokines to mimic tumor microenvironment and stimulate cell proliferation prior to STP-B treatment (100 nM) for 24 h. Change in cell cycle distribution was calculated. Paired t test was used. **P<0.01. (C) Cytotoxic activity of STP-B was evaluated at 24, 48 and 72 h by Annexin-V staining in 3 MCL cell lines. Graphs represent 4 independent experiments.

Figure 5.

CTPS1 inhibition synergizes with BCL2 inhibition in vitro and in vivo. (A) Bliss synergy scores were determined after treatment for 72 hours (h) with STP-B/venetoclax (ven) or STP-B/ibrutinib (ibru) combinations in mantle cell lymphoma (MCL) cell lines. Detailed results are shown in Online Supplementary Figure S5. (B) BCL2 dependence following STP-B treatment was assessed by BH3 profiling in Z138. Cells were pretreated for 24 h with 50 or 500 nM STP-B. Cytochrome C release was analyzed after treatment with 10 or 20 μM of ven (BCL2-i) as indicated in the Online Supplementary Appendix. Graph represents 4 independent experiments. Paired t test was used. *P<0.05, **P<0.01. (C) Immunoblotting of anti-apoptotic BCL2 family members was performed in Z138 and JeKo-1 treated with STP-B at 50 and 500 nM for 24 h. Protein levels normalized to GAPDH level are indicated. (D) Puromycin incorporation assay was performed to directly evaluate the rate of protein synthesis upon STP-B treatment. Cells were pretreated for 24 h with 50 or 500 nM STP-B, prior to puromycin addition as indicated in the Online Supplementary Appendix. Graph indicates the percentage of puromycin incorporated. (E) The efficacy of STP-B/ven combination was evaluated in vivo using a Z138 xenograft model (N=5 mice per group). STP-B was dosed at 30 mg/kg days 1-4 of a 7-day cycle and ven was dosed at 75 mg/kg days 2-5 of a 7-day cycle for 3 cycles. Statistical analysis was made using a two-way ANOVA test followed by a Tukey’s multiple comparisons test. **P<0.01, ****P<0.0001. subcut.: subcutaneous.