Drivers of de novo Serine/Glycine synthesis in acute leukemia

Cancer cells hijack metabolic pathways in order to provide themselves with building blocks to support their proliferation and survival. Upregulation and addiction to de novo serine/glycine synthesis is an example of metabolic rewiring in cancer cells whereby serine and glycine are synthesised via a side branch of glycolysis. In this review, we focus on upregulation of endogenous serine/glycine production in acute leukemia, namely T‐cell acute leukemia (T‐ALL) and acute myeloid leukemia (AML). Several genetic lesions directly driving the serine/glycine addiction in acute leukemia have been established. Additionally, indirect regulation of de novo serine/glycine synthesis is observed in acute leukemia.


Introduction
Whereas most cells take up serine and glycine from their environment, approximately 30% of all cancers activate the de novo serine/glycine synthesis pathway (SSP) in order to support their proliferation and survival (1). Serine/glycine, produced via a side branch of glycolysis, provides cancer cells with an antioxidative capacity and building blocks to synthesize purines. Furthermore, it regulates DNA methylation and lipid metabolism (2)(3)(4). Besides copy number gains of the serine/glycine enzyme genes (5,6), also oncogenes and tumour suppressors upregulate the SSP. In this review, we focus on the reprogramming of serine/glycine metabolism and the corresponding drivers in acute leukemias, a group of malignant hematopoietic disorders with uncontrolled proliferation of bone marrow precursors.

SSP hyperactivation in acute lymphoblastic leukemia (ALL)
Hyperactivation of the SSP is observed in T-cell ALL (T-ALL), with elevated PSPH, PHGDH or PSAT1 mRNA expression in more than 70% of patient samples. PSPH knockdown reduces T-ALL cell proliferation and in vivo expansion of leukemia cells (7). SHMT1/2 inhibition causes S/G2 cell cycle arrest and impairs leukemia progression and burden in vivo (8,9). Several genetic defects driving serine/glycine addiction in T-ALL have been discovered. A first example is the ribosomal protein mutation RPL10 R98S, present in 8% of paediatric T-ALL patients (7,10). RPL10 R98S T-ALL cells display enhanced transcription and protein 13 translation of PSPH. C -glucose tracer analysis showed enhanced synthesis of serine and 6 glycine, which the cells utilize for purine synthesis. RPL10 R98S cells secrete excessive serine/glycine, supporting the survival of healthy niche cells that can help to sustain the cancer cells (7). Moreover, RPL10 mutated cells are sensitive to the repurposed dual SHMT1/2 inhibitor sertraline, further confirming the role of RPL10 R98S as a driver of serine/glycine synthesis (11). Upregulation of PSPH, PSAT1 and SHMT1/2 expression also occurs in the NKX2-1 T-ALL subgroup (9,12), where the transcription factor NKX2-1 is ectopically expressed due to NKX2-1 rearrangements (13)(14)(15). NKX2-1 directly binds the PHGDH, PSAT1, PSPH and SHMT2 promoter and PSPH enhancer, inducing expression of these genes. As a result, NKX2-1 knockout in RPMI-8402 T-ALL cells causes 80% reduction of serine/glycine enzyme RNA expression and reduces de novo serine production. These knockout cells also suffer from reduction of purine nucleotides and redox molecules (glutathione, GSH), resulting in enhanced intracellular ROS levels. Across a panel of >10 T-ALL cell lines, the NKX2-1 expressing RPMI-8402 leukemic cells show the highest sensitivity to sertraline. Sertraline impairs disease progression in mice xenografted with human patient derived NKX2-1 positive T-ALL. Strikingly, an even stronger reduction in disease progression is obtained by combining sertraline with a serine/glycine free diet in these mice, further underscoring the dependency of NKX2-1 cancer cells on serine/glycine synthesis (12). Interestingly, also indirect induction of the SSP has been observed in T-ALL. In this context, p15 and p16, inhibitors of the CDK4/6 complex (16), are deleted in the large majority of T-ALL patients (17,18). The resulting high expression of the CycD3/CDK6 complex causes inactivation of glycolysis enzymes PFKP and PKM2. This re-directs the glycolysis intermediates into the SSP, supplying the tumour cells with antioxidants GSH and NADPH (19). Gain-of-function mutations in NOTCH1 are present in 60% of T-ALL patients (20) and directly induce C-MYC mRNA expression (21). C-MYC upregulates serine/glycine synthesis enzyme transcription (22,23) leading to increased GSH and nucleotide levels (23). Enhanced expression of the histone methyltransferase EHMT2 is present in T-ALL patients (24). EHMT2 upregulates transcription of the SSP genes by H3K9 mono-methylation leading to enhanced de novo serine/glycine synthesis (25). Interestingly, increased EHMT2 expression and H3K9me1 levels are also seen in acute myeloid leukemia (AML) (26,27). Additionally, in T-ALL cells EHMT2 suppresses p53 levels (24), a known suppressor of PHGDH expression and the SSP in melanoma (28), which is also targeted by mutations in T-ALL (29).

SSP hyperactivation in acute myeloid leukemia (AML)
AML patient samples display enhanced expression of SHMT1, and PHGDH expression is a prognostic factor for overall survival (30,31). Increased ATP levels in the bone marrow niche of mice can trigger transcription factor CREB1 to enhance PHGDH expression and maintain serine metabolism in AML (32). Also glutamine withdrawal leads to PHGDH and PSAT1 upregulation (33), likely due to lower GSH levels and resulting in oxidative stress and ATF4 activation (34). Interestingly, also ATF3 can directly bind and regulate enzymatic serine/glycine synthesis genes in AML (35,36). Internal tandem duplications in tyrosine kinase FLT3 (FLT3-ITD), present in 30% of AML patients, promote the SSP through the mTORC1-ATF4 axis, whereby ATF4 binds the PHGDH, PSAT1, SLC1A4 and SLC1A5 promoter or enhancer regions. FLT3-ITD positive patients have higher expression of PHGDH, PSPH and SHMT1. Additionally, bone marrow cells from an MLL-rearranged, inducible FLT3-ITD-driven AML mouse model, express elevated levels of Phgdh, Psat1 and serine transporters (Slc1a4 and Slc1a5). FLT3 inhibition by quizartinib reduces intracellular serine/glycine levels in FLT3-ITD cells, leading to decreased synthesis of ATP and antioxidant GSH. CRISPR-Cas9 mediated PHGDH depletion and pharmacological inhibition by WQ-2101 induces apoptosis and eradicates FLT3-ITD cells in vitro and in vivo (34).
Mutations in DNMT3A occur in approximately 20% of AML patients and have been associated with inferior prognosis. DNMT3A R882H AML cells and patients display enhanced expression of PSPH and PSAT1. In addition, elevated intracellular GSH levels promote the proliferation of these mutant cells (37), which suggests that DNMT3A R882H may be an additional driver of SSP.

Conclusion
In conclusion, several drivers rendering acute leukemia cells addicted to upregulated cellular serine/glycine production have been discovered. These recent findings led to the discovery of clinically usable inhibitors, such as the SHMT1/2 inhibitor sertraline, which can be used for targeted therapies inhibiting the SSP in leukemia patients.