Background

Multiple myeloma (MM) is characterized by proliferation of malignant antibody-producing B cells known as plasma cells. These malignant plasma cells accumulate in the bone marrow and often secrete abnormal antibodies, resulting in pathological bone fractures, renal failure, infections and bone marrow failure. Despite impressive progress in treatments achieved over recent years, the disease remains largely incurable¹.

Fast proliferating cells have a huge demand for cellular building blocks such as pyrimidines. Cells have two possibilities to generate pyrimidines. Either pyrimidines are recycled from existing cell constituents via the salvage pathway or they are synthesized de novo from small molecule precursors. While resting cells can cover their pyrimidine requirements using the salvage pathway, fast proliferating cells such as cancer cells rely on the de novo synthesis of pyrimidines. Cytidine triphosphate synthase is the rate limiting step in the de novo synthesis pathway and is catalyzed by two isoforms, CTPS1 and CTPS2².

It was previously reported that inherited homozygous mutation of CTPS1 results in severe childhood immunodeficiency, due to a failure of B and T cells to proliferate following antigen challenge. Interestingly, no phenotype was seen in these individuals outside of the blood system^3,4. This observation, along with the fact that targeting DNA and RNA synthesis has proven to be a successful treatment strategy in a wide variety of cancers, prompted us to explore CTPS1 as a potential therapeutic target in MM, as a means to kill malignant plasma cells while sparing non-hematopoietic tissues.

Results

Our investigations began with the exploration of CTPS1 expression in publicly available MM data sets, which revealed a strong correlation between higher CTPS1 expression and advanced or poor risk disease. Additionally, our analysis of DepMap CRISPR knockout data confirmed the dependence of MM cell lines on the de novo pyrimidine biosynthesis, with the salvage pathway appearing redundant.

To further validate CTPS1 as a potential target, we initiated in vitro experiments with CTPS1 knockout cell lines. As anticipated, these cell lines failed to proliferate and underwent apoptosis, highlighting the importance of CTPS1 to MM cell survival.

Recognizing the potential of CTPS1 as a target, we went on to assess the impact of CTPS1 loss in a broad spectrum of MM cell lines. For these experiments, we used a novel and highly-selective inhibitor of CTPS1 which demonstrated more than a 1,300 fold selectivity for inhibition of CTPS1 over CTPS2. This inhibitor was tested in 12 MM cell lines, revealing anti-proliferative effects in 6 out of 12 cell lines after 72 hours exposure. These effects were accompanied by apoptosis induction, mirroring our findings in CTPS1 knockout cells. Subsequent assessment of the CTPS1 inhibitor in a xenograft model confirmed the ability of selective CTPS1 inhibition to block tumor growth.

To understand the underlying molecular consequences of CTPS1 inhibition, we focused on cell cycle regulation and the DNA damage response (DDR) pathway, both known to be impacted by nucleotide deficiency. Indeed, our analyses revealed that CTPS1 inhibition resulted in S phase arrest, reduced histone 3 phosphorylation (indicating reduced entry into mitosis), activation of key DDR pathway intermediaries (CHEK1, CHEK2) and induction of DNA damage.

We also investigated the 6 cell lines that showed no response to pharmacological CTPS1 inhibition in the 72 hour viability assays. Despite this initial apparent lack of effect, CTPS1 inhibition in these cell lines exhibited molecular consequences similar to sensitive lines, including S phase arrest, decreased histone 3 phosphorylation, DDR pathway activation and DNA damage. This challenged the notion of ‘resistance’ and led us to explore these cell lines in more details. We observed that cell lines with shorter doubling times are more sensitive to CTPS1 inhibition, prompting investigation into whether the duration of the experiments impacted the outcomes. Indeed, extended cell culture over 7 days resulted in profound growth inhibition, and induction of apoptosis in some initially resistant cell lines.

Given the observed activation of the DDR pathway following CTPS1 inhibition, including in cell lines that initially appeared resistant, we hypothesized that CTPS1 inhibition might show synergistic activity with DDR pathway inhibitors. Thus, we combined CTPS1 inhibition with inhibitors of ATR (ceralasertib), CHEK1 (rabusertib) or WEE1 (adavosertib). As anticipated, strong synergy was observed with all these combinations, resulting in early onset of apoptosis, even in initially resistant cell lines.

Where do we go from here?

In these studies, we identify CTPS1 as a novel target which could add a new precision weapon to the MM therapeutic armory. Importantly, the mechanisms underlying the efficacy of CTPS1 inhibition in MM relate to basic cellular process including DNA replication, cell cycle regulation and DNA damage sensing. As such, we expect that our results should be transferable to other types of cancer, including both hematological and solid tumors. 

Looking forward, there are several issues that could be addressed by future studies. The mechanisms by which some cancer cells are able to tolerate pyrimidine depletion for longer periods of time remains to be fully elucidated, as does the apparent ability of these cells to tolerate higher levels of DNA damage. Therapy for hematological cancers is moving away from chemotherapy to combinations of targeted agents, with MM serving as a paradigm. Additional exploration of drug combinations based around CTPS1 inhibition would further this cause, along with assessment of the tolerability of these combinations in preclinical models. Finally, exploration of CTPS1 inhibition in solid tumors is warranted, given the fundamental cellular processes that are disrupted by CTPS1 inhibition in MM cells.

A selective CTPS1 inhibitor entered clinical development at the end of 2022 for patients with relapsed/refractory B or T cell lymphoma (NCT05463263).

References

1. Zanwar S, Kumar S. Disease heterogeneity, prognostication and the role of targeted therapy in  multiple myeloma. Leuk Lymphoma 2021; 62: 3087–3097.
2. Wang W, Cui J, Ma H, Lu W, Huang J. Targeting Pyrimidine Metabolism in the Era of Precision Cancer Medicine. Front Oncol. 2021;11:684961. 
3. Martin E, Palmic N, Sanquer S, Lenoir C, Hauck F, Mongellaz C et al. CTP synthase 1 deficiency in humans reveals its central role in lymphocyte proliferation. Nature 2014; 510: 288.
4. Martin E, Minet N, Boschat A-C, Sanquer S, Sobrino S, Lenoir C et al. Impaired lymphocyte function and differentiation in CTPS1-deficient patients result  from a hypomorphic homozygous mutation. JCI insight 2020; 5.