Transcriptional Repression of DAPK1 Characterizes a Resistant Phenotype of AML Enforced by Flt3 Signaling and Exclusive Nuclear Abundance of Non-Canonical NFkB2/p52: Synergistic Activity for Flt3 Inhibition along with HDAC Inhibition, or by NFkB Inhibition, for Potentiating ER Stress Apoptosis.
Authors: Shanmugam Rajasubramaniam , Wilson-Weekes Annique , Sayar Hamid , Suvannasankha Attaya , Cardoso Angelo A. , Sargent Katie J , Cripe Larry D. , Kalvakolanu Dhananjaya V , Boswell H. Scott 
Published: October 2019

Abstract
Abstract 2387
Poster Board II-364
Treatment failure in AML has been linked with the consequences of Flt3 signaling, although effectors of such resistance are uncertain. However, poor prognostic Flt3-ITD-containing AML's and some other intermediate prognostic groups have been found to have blunted spontaneous unfolded protein response/ER stress apoptotic pathway (Schardt, et al. Clin Canc Res. 15: 3834, 2009) that correlates with adverse outcome of therapy. We investigated the origins of suppressed ER-stress apoptosis in poor-risk AML by assaying a rate-limiting enzyme effector, DAPK1. Real-time RT-PCR analysis of DAPK1 expression, as well as its putative transcriptional activator(s), including c-jun, and its transcriptional repressor(s), including relB, and a gene-set enrichment array (GSEA) of NFkB- and c-jun-responsive (cytokine) genes, was undertaken in 30 AML samples. This included a control cohort of good-prognosis CBF+ve patients. A group of poor prognosis patients, especially those with normal karyotype and Flt3 (ITD) mutation, and those with MLL translocations, had on-average 10-fold (range 2-100-fold) reduction of DAPK1 transcripts, when normalized to expression of c-jun, a transcriptional activator of DAPK1, or to Meis1, another c-jun transcriptional target, which is a validated AML adverse prognostic array gene. Indeed, NK Flt3-ITD or t-MLL AML blasts with DAPK1 repression were characterized by exclusive nuclear presence of p52NFkB/relB, known to attract epigenetic and repressive components, including histone deacetylase enzymes (HDAC's) to the tandem CRE/NFkB sites (“integrated circuit”) at 5' DAPK1 -177bp and -134bp, respectively (Puto and Reed, Genes&Dev 22:998, 2008; Huang et al. Mol Cell 35: 48, 2009; Gade et al. Mol Cell Biol. 28: 2528, 2008). In the t-MLL/Flt3-ITD AML cell line MV-4-11, knockdown of NIK, IKK1, or p52NFkB2, while sparing Flt3-to-JNK/c-jun signaling, sharply upregulated (3-5-fold) DAPK1 expression. Flt3 inhibition in primary AML cells by using R406 (Rigel Pharmaceuticals, also a lead compound in CLL/NHL) a selective tyrosine kinase inhibitor that affects Flt3 auto-phosphorylation (and syk with similar cellular IC50's) also de-repressed DAPK1 expression in AML blasts. This outcome was associated with an inhibition of NIK and p52NFkB (both were also inhibited by Flt3 knockdown in MV-4-11) and a rise in apoptosis in R406-treated primary AML cells occurred. The combination of R406 with an HDAC inhibitor, suberoyl bis-hydroxamic acid (SBHA) showed synergistic inhibition of proliferation (tritiated thymidine incorporation). ER stress pathway activation evidenced by IREalpha and p-eIF2alpha upregulation was enhanced in primary AML blasts. Bortezomib (a proteasome-targeted inhibitor of NFkB/ER stress potentiator) also strongly inhibited p52NFkB activity and induced apoptosis. These studies reveal that transcriptional/epigenetic suppression of DAPK1 in poor-risk AML is enforced by a Flt3-to-NIK-to-p52NFkB2/relB signal cascade, and support a causative influence on clinical outcomes. Thus, resistance is subject to inactivation by a Flt3-selective TKI, in combination with HDAC inhibitor/or by proteasome-NFkB inhibitor with consequent ER stress apoptosis. A potential subsidiary role for syk inhibition downstream from Flt3 also exists. Taken together, these maneuvers reverse the resistance to apoptosis of NK Flt3-ITD or tMLL primary AML blasts, and including others with over-expressed/active Flt3 wild-type.

Disclosures:
No relevant conflicts of interest to declare.