Introduction: The ability to reprogram cellular metabolism including enhanced de novo nucleotide synthesis is a hallmark of cancer and essential for tumor progression and survival. The pyrimidine synthesis pathway has been proposed as a metabolic sensitivity for cancer and over the past few decades several inhibitors of enzymes involved in the pathway have been evaluated in clinical trials (e.g. avicin, CPEC, PALA, leflunomide) primarily in solid tumors with limited efficacy and unacceptable toxicity. The final step in the pyrimidine de novo nucleotide synthesis pathway is the conversion of UTP to CTP. This step is unusual in the pathway in having two enzymes which catalyze the same reaction, CTP synthase 1 (CTPS1) and CTPS2, which have the same activity but very different cell type dependencies. CTPS1 has been shown to have an essential and non-redundant role in lymphocyte proliferation with the identification of an ultra-rare human hypomorphic mutation resulting in an 85% reduction in CTPS1 activity which manifests as a profound effect upon lymphoid cells but with no effects on other cell types. This hypomorphic mutation prevents proliferation of T-cells and B-cells following activation, thus selective inhibition of CTPS1 represents a novel targeted approach to inhibit proliferation of lymphocytes in hematological malignancies without inhibiting CTP synthesis in other cell types which utilise CTPS2. We have identified and optimized orally bioavailable selective inhibitors of CTPS1 (exemplified by STP938) and hypothesized that STP938 would demonstrate anti-tumor effects on cells derived from hematological malignancies.
Methods: The in vitro anti-tumor activity of selective CTPS1 inhibition was investigated in a 2D monolayer assay in a panel of 200 cancer cell lines. Cell viability was measured with the Cell Titer-Blue Cell Viability Assay after 4 days incubation. Human PBMCs were isolated and T-cells were activated with anti CD3/CD28 antibodies to assess effects of CTPS1 inhibition on intracellular nucleotide levels which were quantified using Liquid Chromatography Mass Spectrometry (LC-MS). Jurkat T-ALL cells and WI38 embryonic fibroblasts were used to assess apoptosis which was quantified using the Caspase-Glo 3/7 Assay. Murine xenograft studies were conducted in NOD.SCID mice implanted with T-ALL cell lines to assess anti-tumor effects of CTPS1 inhibitors.
Results: The panel of cancer cell lines consisted of 56 derived from hematological cancers and 141 derived from solid tumors. STP938, a potent and selective inhibitor of CTPS1 specifically blocked cell proliferation of the hematological cancers. 43 (77%) of the hematological cell lines had an IC50 <100nM whereas only 15% of the solid tumor derived cell lines were affected by the CTPS1 inhibitor. The T-cell derived cell lines were extremely sensitive with many IC50 <10nM. Profiling of intracellular nucleotide levels using activated human T-cells exposed to increasing concentrations of STP938 demonstrated the selective depletion of CTP but not ATP or GTP (see Figure). Jurkat T-ALL cells and WI38 fibroblasts were evaluated for markers of apoptosis in the presence of increasing concentrations of STP938. The Jurkat T-ALL cells exhibited a dose dependent increase in caspase 3/7 levels indicative of apoptosis. The WI38-fibroblast cells were unaffected. Murine xenograft studies using NHL T lymphoma and leukemia cell lines were conducted with oral dosing of CTPS1 inhibitor starting at the time of tumor cell inoculation or once tumors were established. In all cases, in a dose-dependent manner, CTPS1 inhibitors reduced or ablated tumor growth meeting NCI criteria for efficacy.
Conclusions: Our preliminary results indicate that inhibition of CTPS1 enables cell specific inhibition of de novo pyrimidine nucleotide synthesis with a particular sensitivity displayed by cell lines derived from hematological malignancies. This disruption of de novo nucleotide synthesis by CTPS1 inhibition selectively induces apoptosis in lymphocyte cells but not in other cell types that are able to rely upon CTPS2 for their CTP synthesis. Orally bioavailable small molecule inhibitors of CTPS1 exert an anti-tumor effect in vivo in murine xenograft models and thus inhibition of CTPS1 represents a novel targeted approach to treat hematological malignancies.
Parker:Step Pharma: Current Employment.
Asterisk with author names denotes non-ASH members.