SQSTM1/p62 Is a Necessary Cofactor In MDS/AML With Deletion Of Mir-146a
Authors: Fang Jing , Barker Brenden , Bolanos Lyndsey , Liu Xiaona , Jerez Andres , Makishima Hideki , Rao Dinesh S. , Mulloy James C. , Maciejewski Jaroslaw P. , Cancelas Jose A. , Starczynowski Daniel T 
Published: October 2019

Abstract
Deletions involving chromosome 5 (del(5q)) are the most common genetic abnormalities in Myelodysplastic Syndrome (MDS) and secondary Acute Myeloid Leukemia (AML). Chromosome 5q deletions extending beyond q34 portend a worse overall survival, are associated with high-risk (HR) disease, and exhibit significant downregulation of miR-146a, a gene residing on the extended deleted region on 5q34. Additional evidence linking miR-146a loss to HR del(5q) MDS/AML comes from mouse genetic studies; miR-146a-/- mice develop a myeloid proliferative disease and myeloid tumors, in part by derepression of TNFR associated factor 6 (TRAF6) and persistent NF-kB activation. To determine the contribution of miR-146a deficiency to HR MDS/AML, we first examined hematopoietic stem/progenitor cells (HSPC) from miR-146a-/- mice. miR-146a-/- HSPC are highly proliferative, and exhibit increased cell survival and altered HSC fitness. In addition, genetic and/or pharmacologic inhibition of TRAF6/NF-kB signaling impairs cell cycle progression and preferentially leads to apoptosis of malignant miR-146a-/- HSPC. Although inhibiting the TRAF6/NF-kB axis may represent a therapeutic opportunity in miR-146alow MDS/AML patients, unfortunately, NF-kB inhibitors in clinical trials have been disappointing and ones for TRAF6 do not exist. Chromosome deletions that target tumor suppressor genes also involve multiple neighboring genes, such as with del(5q), and loss of certain neighboring genes may expose cancer-specific vulnerabilities. To overcome the limitations of NF-kB inhibitors and identify novel therapeutic targets, we examined the expression of all genes residing within the long arm on chr 5 (5q11-q35) from del(5q) MDS and control CD34+ cells and built molecular networks using GeneConnector functionality in NetWalker. A single major intrachromosomal NF-kB signaling node formed corresponding to the overexpressed chr 5q genes. Among the compensated/overexpressed genes residing on chr 5q and within the NF-kB node, SQSTM1/p62 (5q35) emerged as an obvious candidate as it is an essential cofactor for NF-kB activation by binding TRAF6. First, we evaluated the contribution of p62 to the malignant miR-146alow HSPC phenotype. Overexpression of p62 enhanced proliferation of miR-146a-/- HSPC by promoting G2/M cell cycle progression. Conversely, knockdown of p62 in miR-146a-/- HSPC led to cell cycle arrest and rescued defective myeloid engraftment in competitive HSC transplantation assays, suggesting p62 is required in miR-146alow leukemic cells. Furthermore, the importance of p62 was confirmed in MDS/AML cell lines and patient samples. RNAi-mediated knockdown of p62 resulted in a G2/M cell cycle arrest, reduced cell survival, and impaired leukemic progenitor function, underscoring the importance of p62 in MDS/AML. In addition, interfering with p62-TRAF6 binding by overexpressing a small peptide corresponding to the p62-TRAF6 binding interface suppressed TRAF6-mediated NF-kB activation, and similarly inhibited cell cycle progression and induced apoptosis of human miR-146alow leukemic cells. Collectively, these findings reveal an intrachromosomal gene network that not only drives HR del(5q) myeloid malignancies, but also exposes them to cancer-specific therapeutic vulnerability by disrupting the binding between p62 and TRAF6.

Disclosures:
Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.