Lncrna Morrbid Regulates Myeloid Cell Survival of AML Driven By a Combination of FLT3ITD and Epigenetic Mutations

FLT3ITD mutations are observed in ~30% of all acute myeloid leukemia (AML) patients, primarily classified into M4/M5 subtype, and associated with poor survival. Frequently, FLT3ITD mutations are co-expressed with epigenetic mutations including TET2 and DNMT3A and portend a poor prognosis. We analyzed human RNA sequencing datasets for lncRNAs expressed in myeloid malignancies and identified novel lncRNAs including MORRBID. MORRBID is located close to BCL2L11, encoding the pro-apoptotic protein BIM. Human TCGA analysis showed MORRBID overexpression in AML patients with TET2 mutations and its association with poor overall survival. Our independent study using over 100 patients showed that AML patients with FLT3ITD mutation express significantly higher MORRBID compared to patients without such mutation. Consistently, mice lacking Tet2 or Dnmt3a but expressing Flt3ITD develop AML and express higher levels of Morrbid in Lin- Sca-1+ Kit+ (LSK) cells. We hypothesized that MORRBID may not only act as a novel prognostic and diagnostic biomarker for AML but also as a therapeutic target in AML bearing a combination of Tet2^-/-;FLT3^ITD/ITD (TF) and Dnmt3a^-/-;FLT3^ITD/ITD (DF) mutations. We generated Tet2^-/-;Flt3^ITD/ITD;Morrbid^-/-(TFM)and Dnmt3a^-/-;FLT3^ITD/ITD;Morrbid^-/-(DFM)mice and assessed their hematologic parameters. Loss of Morrbid in the context of TF and DF rescued majority of peripheral blood (PB) abnormalities associated with these models of AML including increased myeloid cells (neutrophils and monocytes). In addition, the infiltration of leukemia cells in lung was significantly removed in the setting of Morrbid loss. Mechanistically, a significant reduction in the expression of Bim was observed in Lin- cells derived from TF and DF mice, which was associated with increased expression of Morrbid. However, Morrbid loss increased expression of Bim, induced apoptosis and reversed the enhanced survival seen in LSK cells derived from TF and DF AML mice. Overall, the increased bone marrow (BM) cellularity observed in TF primary mice was normalized to WT levels in the absence of Morrbid, which was associated with an increase in apoptosis in both BM myeloid progenitors as well as in more mature myeloid cells. To determine if in a competitive setting, loss of Morrbid in the setting of TF leukemia stem cells would alter clonal hematopoiesis, overall engraftment and AML development, we performed a competitive transplant assay and observed complete correction in term of abnormal leukemia cell counts, including correction in PB counts, presence of Mac-1⁺ blasts in PB as well as overall engraftment of TFM cells 24 weeks post-transplant. The rescue in these defects associated with TF donor cells in the absence of Morrbid was observed in the PB, BM and spleen. Loss of Morrbid mitigates the clonal expansion of AML donor cells in both primary and secondary competitive transplant assays. Transplantation of whole BM cells bearing TFM cells resulted in a partial correction of the AML phenotype including correction of mature myeloid cells in PB and BM of the recipients. Importantly, the absolute number of leukemia progenitor cells, which are normally increased in the BM with TF donors, were also restored to WT levels in recipient mice with TFM donors. Finally, the reduced overall survival defects seen upon secondary transplantation of TF cells was significantly prolonged in the absence of Morrbid. Our results warrant that MORRBID is a novel prognostic/diagnostic biomarker and a therapeutic target for AML.