REZI-20-037-1

Serine/one-carbon metabolism provides critical resources for nucleotide biosynthesis and epigenetic maintenance and is thus necessary in cancer cell growth, although the detailed regulatory mechanisms remain unclear. We uncover a critical role of glycogen synthase kinase 3 (GSK3) in regulating the expression of serine/one-carbon metabolic enzymes. Nuclear enrichment of GSK3 significantly suppresses genes that mediate de novo serine synthesis, including PHGDH, PSAT1, PSPH, and one-carbon metabolism, including SHMT2 and MTHFD2. FRAT1 promotes nuclear exclusion of GSK3, enhances serine/one-carbon metabolism, and, as a result, confers cell vulnerability to inhibitors that target this metabolic process such as SHIN1, a specific SHMT1/2 inhibitor. Furthermore, pharmacological or genetic suppression of GSK3 promotes serine/one-carbon metabolism and exhibits a significant synergistic effect in combination with SHIN1 in suppressing cancer cell proliferation in cultured cells and in vivo. Our observations indicate that inhibition of nuclear GSK3 signaling creates a vulnerability, which results in enhanced efficacy of serine/one-carbon metabolism inhibitors for the treatment of cancer.

Increasing fetal hemoglobin (HbF) provides clinical benefit in patients with sickle cell disease (SCD). We recently identified heme-regulated inhibitor (HRI, EIF2AK1), as a novel HbF regulator. Because HRI is an erythroid-specific protein kinase, it presents a potential target for pharmacologic intervention. We found that maximal HbF induction required >80% to 85% HRI depletion. Because it remains unclear whether this degree of HRI inhibition can be achieved pharmacologically, we explored whether HRI knockdown can be combined with pharmacologic HbF inducers to achieve greater HbF production and minimize potential adverse effects associated with treatments. Strongly cooperative HbF induction was observed when HRI depletion was combined with exposure to pomalidomide or the EHMT1/2 inhibitor UNC0638, but not to hydroxyurea. Mechanistically, reduction in the levels of the HbF repressor BCL11A reflected the cooperativity of HRI loss and pomalidomide treatment, whereas UNC0638 did not modulate BCL11A levels. In conjunction with HRI loss, pomalidomide maintained its HbF-inducing activity at 10-fold lower concentrations, in which condition there were minimal observed detrimental effects on erythroid cell maturation and viability, as well as fewer alterations in the erythroid transcriptome. When tested in cells from patients with SCD, combining HRI depletion with pomalidomide or UNC0638 achieved up to 50% to 60% HbF and 45% to 50% HbF, respectively, as measured by high-performance liquid chromatography, and markedly counteracted cell sickling. In summary, this study provides a foundation for the exploration of combining future small-molecule HRI inhibitors with additional pharmacologic HbF inducers to maximize HbF production and preserve erythroid cell functionality for the treatment of SCD and other hemoglobinopathies.