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Infants diagnosed with KMT2A-rearranged (KMT2Ar) acute lymphoblastic leukemia (ALL) have a poor prognosis with an event free survival of 23-44%. To identify new treatment approaches we previously performed in vitro and in vivo assays to evaluate the activity of FDA approved compounds in 15 primary KMT2Ar infant leukemia samples. Three classes of agents were found to be active in these assays: proteasome inhibitors, anthracyclines, and histone deacetylase inhibitors (HDACi). KMT2Ar infant leukemia samples were exquisitely sensitive to the proteasome inhibitor bortezomib, requiring 10-100 fold less drug to achieve 50% toxicity when compared to non-KMT2Ar childhood ALL. Bortezomib is FDA approved for multiple myeloma and laboratory studies using this model system have previously demonstrated responses to be mediated through several mechanisms including NFKB inhibition, stabilization of cell cycle regulatory proteins, and perhaps most importantly the induction of an unfolded protein response (UPR) and endoplasmic reticulum (ER)-stress-induced apoptosis. To evaluate global protein dynamics in KMT2Ar ALL cells treated with bortezomib, we performed tandem mass tag (TMT) quantitative mass spectrometry on synchronized SEM cells exposed to either 50nM of bortezomib or DMSO at 0 hours (hr), 6hr, 12hr, 16hr, and 20hr. Applying pairwise comparison for 9232 unique proteins measured over the time course compared to untreated controls, we identified 1593 proteins with a log2 fold change >1.5 in bortezomib treated cells compared to 101 proteins in the DMSO control (FDR While GADD34 has been shown to be the main phosphatase that functions in a negative feedback loop to resolve cell stress, recent data suggests that stabilization of CReP mRNA by ER stress is able to reverse eIF2α phosphorylation at later stages of UPR leading to re-expression of key UPR proteins. Further, p-eIF2α-attenuated protein synthesis, and not ATF4 mRNA translation has been shown to promote cell survival. Our data support a model whereby the UPR and ER-stress in KMT2Ar ALL cells is induced upon exposure to bortezomib leading to increased levels of ATF4 and CHOP. Attenuation of p-eIF2α by CReP further contributes to cell death through the recovery of protein synthesis in a setting of limited protein folding capacity. These results support the use of proteasome inhibitors in KMT2Ar leukemia which is currently being formally evaluated in a Phase II clinical trial for newly diagnosed patients with infant ALL (NCT02553460). Disclosures Gruber: Bristol-Myers Squibb: Consultancy.

Growth factor independence 1 (Gfi-1) is a part of the transcriptional network that regulates the development of adult hematopoietic stem and progenitor cells. Gfi-1-null (Gfi-1(-/-)) mice have reduced numbers of hematopoietic stem cells (HSCs), impaired radioprotective function of hematopoietic progenitor cells (HPCs), and myeloid and erythroid hyperplasia. We found that the development of HPCs and erythropoiesis, but not HSC function, was rescued by reducing the expression of inhibitor of DNA-binding protein 2 (Id2) in Gfi-1(-/-) mice. Analysis of Gfi-1(-/-);Id2(+/-) mice revealed that short-term HSCs, common myeloid progenitors (CMPs), erythroid burst-forming units, colony-forming units in spleen, and more differentiated red cells were partially restored by reducing Id2 levels in Gfi-1(-/-) mice. Moreover, short-term reconstituting cells, and, to a greater extent, CMP and megakaryocyte-erythroid progenitor development, and red blood cell production (anemia) were rescued in mice transplanted with Gfi-1(-/-);Id2(+/-) bone marrow cells (BMCs) in comparison with Gfi-1(-/-) BMCs. Reduction of Id2 expression in Gfi-1(-/-) mice increased the expression of Gata1, Eklf, and EpoR, which are required for proper erythropoiesis. Reducing the levels of other Id family members (Id1 and Id3) in Gfi-1(-/-) mice did not rescue impaired HPC function or erythropoiesis. These data provide new evidence that Gfi-1 is linked to the erythroid gene regulatory network by repressing Id2 expression.

The survival, self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPC) are tightly regulated by extrinsic signals, and intrinsically by transcription factors and their regulatory networks. The molecular and cellular mechanisms, which regulate the complex process of hematopoiesis, depend upon the correct expression of transcription factors and their regulators. One such family of regulators is the inhibitor of DNA binding/differentiation (Id), which is helix-loop-helix proteins that function by acting as dominant negative regulators of transcription factors such as E proteins, ETS, Pax, and retinoblastoma proteins. Expression of Id2, one of the Id family proteins, is regulated by growth factor independence-1 (Gfi-1) encoding a transcriptional repressor. Gfi-1 is required for the development of multiple cell lineages including HSPC and ultimately differentiated blood cells. Although genes have been identified to mediate hematopoietic defects observed in Gfi-1 knockout (Gfi-1 KO) mice including the maturational and developmental defects in granulocyte (CSF-1, RasGRP1, and PU.1) and B cell (PU.1 or Id2), and myeloid hyperplasia (Id2 or HoxA9), Gfi-1-target genes that mediate the defects in radioprotection, maintenance of HSC, and erythroid hyperplasia in Gfi-1 KO mice are unknown. Since Id2 expression is elevated in HSPC of Gfi-1 KO mice and Id2 promotes cell proliferation, we hypothesized that lowering Id2 expression could rescue the HSPC defects in the Gfi-1 KO mice. By transplanting Gfi-1 KO mouse bone marrow cells (BMC) into lethally-irradiated recipient mice, we observed that short-term reconstituting cell (STRC) activity in Gfi-1 KO BMC is rescued by transplanting Gfi-1 KO; Id2 Het (heterozygosity at the Id2 locus) BMC, while the long-term reconstitution defect of HSC was not. Interestingly, lineage- Sca-1- c-Kithi HPC, which enriched for megakaryocyte-erythroid progenitor (MEP) as one of the STRC, were fully restored in mice transplanted with Gfi-1 KO; Id2 Het BMC, in contrast to lack of the HPC in Gfi-1 KO BM-transplanted mice. The restoration of donor c-Kithi HPC was directly correlated with increased red blood cell (RBC) levels in recipient mice, which was produced after donor BM engraftment. Furthermore, we identified that reduced Id2 levels restore erythroid cell development by rescuing short-term hematopoietic stem cell, common myeloid progenitor and MEP in the Gfi-1 KO mice. In addition, burst forming unit-erythroid (BFU-E) colony assay showed that hemoglobinized BFU-E development was restored in Gfi-1 KO BM and spleen by lowering Id2 levels. Unlike Id2 reduction, reducing other Id family (Id1 or Id3) levels in Gfi-1 KO mice does not rescue the impaired development of erythroid and other hematopoietic lineages including myeloid, T and B cells. Abnormal expansion of CD71+ Ter119-/low erythroid progenitor cells was rescued in Gfi-1 KO; Id2 Het BMC compared to those in Gfi-1 KO mice. Thus, we hypothesized that erythroid development was blocked at the early stage of erythropoiesis due to the ectopic expression of Id2 in Gfi-1 KO mice. Using Id2 promoter-driven YFP reporter mice, we found that Id2 is highly expressed in the CD71+ Ter119-/low erythroid progenitors, and decreases as the cells mature to pro-erythroblasts and erythroblasts, suggesting that repression of Id2 expression is required for proper erythroid differentiation in the later stages. The dramatic changes of Id2 expression during erythroid development support our findings that the overexpression of Id2 in the absence of Gfi-1-mediated transcriptional repression causes impaired erythropoiesis at the early stage. To identify the molecular mechanisms that could account for how reduced Id2 levels rescue erythropoiesis in Gfi-1 KO mice, we compared the expression of genes and proteins in Gfi-1 KO; Id2 Het and Gfi-1 KO BMC. Using microarray, qRT-PCR and western blot, we discovered that reduction of Id2 expression in Gfi-1 KO BMC results in increased expression of Gata1, EKlf, and EpoR genes, which are required for erythropoiesis. However, the expression levels of cell cycle regulators were not altered by lowering Id2 expression in Gfi-1 KO mice. These data suggest a novel molecular mechanism in which Gfi-1 modulates erythropoiesis by repressing the expression of Id2 that reduce the levels of Id2 protein, binding to E2A and inhibiting the formation of E2A/Scl transcription enhancer complex. Disclosures: No relevant conflicts of interest to declare.