IntroductionHox proteins are homeodomain-containing transcription factors (TFs) that play a vital role in establishing body plan during development. In addition to this role in body planning, Hox proteins have been implicated in limb regeneration, wound healing, adipogenesis, and hematopoietic stem cell self-renewal. 1 Hoxa9, in particular, is expressed at high levels in early hematopoietic progenitor cells and promotes stem cell expansion. In contrast, Hoxa9 down-regulation is associated with hematopoietic differentiation. 2,3 In keeping with this role, Hoxa9 knock-out mice show defects in B and T-cell lymphopoiesis and myelopoiesis. 4 HOXA9 has been intensively studied because of its central role in human acute leukemias. [5][6][7][8] Early studies of BXH2 mice, which spontaneously develop acute myeloid leukemia (AML) as a result of endogenous retroviral integration, showed that overexpression of Hoxa9, as a result of integrations at the locus, is one of the most common genetic abnormalities in these leukemias. 9,10 Subsequent gene expression profiling studies showed that HOXA9 is expressed in many AMLs. Of 6817 genes tested, HOXA9 was the single most predictive marker for poor prognosis. 11 Certain subtypes of acute leukemias, particularly those with rearrangements of the mixed lineage leukemia (MLL) gene, show especially high expression of A cluster HOX genes, 5,6,8 which is critical for MLL fusion protein-mediated transformation. 12,13 However, deregulation of Hox expression also appears to play a central role in leukemias without MLL rearrangements, including AMLs associated with the CALM-AF10 translocation, fusions of HOXA9 to the nucleoporin gene NUP98 in a subset of leukemias with the t(7;11), 14,15 overexpression of CDX2 or CDX4 [16][17][18] and identified T-cell acute lymphoblastic leukemia cases with translocations between the TCR␤ and the HOXA9/HOXA10 locus. 19

The transcription factor NF-κB is required for lymphocyte activation and proliferation as well as the survival of certain lymphoma types 1, 2 . Antigen receptor stimulation assembles an NF-κB activating platform containing the scaffold protein CARMA1/CARD11, the adaptor BCL10, and the paracaspase MALT1 (CBM complex), linked to the inhibitor of NF-κB kinase (IKK) complex 3-12 , but signal transduction is not fully understood 1 . We conducted parallel screens involving a mass spectrometry analysis of CARMA1 binding partners and an RNAi screen for growth inhibition of the CBM-dependent "activated B cell-like" (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) 12 . Here, we report that both screens identified casein kinase 1α (CK1α) as a bifunctional regulator of NF-κB. CK1α dynamically associates with the CBM complex upon T cell receptor (TCR) engagement to augment cytokine production and lymphocyte proliferation. However, CK1α kinase activity plays a counterposing role by subsequently promoting the phosphorylation and inactivation of CARMA1. CK1α has thus a dual "gating" function which first promotes and then terminates receptor-induced NF-κB. ABC DLBCL cells required CK1α for constitutive NF-κB activity indicating that CK1α functions as a "conditionally essential malignancy" (CEMal) gene -a member of a new class of potential cancer therapeutic targets.To better understand signal regulation by the CBM complex, we performed a mass spectrometry proteomic screen following CARMA1 immunoprecipitation. Sixteen peptides covering 54% of CK1α were isolated from an excised band ( Fig. 1a and Supplementary Fig. 1). CK1α belongs to the CK1 family of serine/threonine protein kinases, which regulates

HOXA9 is a homeodomain-containing transcription factor that plays an important role in hematopoietic stem cell expansion and is commonly deregulated in acute leukemias. A variety of upstream genetic alterations in acute myeloid leukemia (AML) lead to overexpression of HOXA9, which is a strong predictor of poor prognosis. In many cases, HOXA9 has been shown to be necessary for maintaining leukemic transformation, however the molecular mechanisms through which it promotes leukemogenesis remain elusive. Recent work has established that HOXA9 regulates downstream gene expression through binding at promoter distal enhancers along with a subset of cell-specific cofactor and collaborator proteins. Increasing efforts are being made to identify both the critical cofactors and target genes required for maintaining transformation in HOXA9-overexpressing leukemias. With continued advances in understanding HOXA9-mediated transformation, there is a wealth of opportunity for developing novel therapeutics that would be applicable for the greater than 50% of AML with overexpression of HOXA9.

Purpose of review HOXA9 is a homeodomain transcription factor that plays an essential role in normal hematopoiesis and acute leukemia, where its over expression is strongly correlated with poor prognosis. This review highlights recent advances in the understanding of genetic alterations leading to deregulation of HOXA9 and the downstream mechanisms of HOXA9-mediated transformation. Recent findings A variety of genetic alterations including MLL-translocations, NUP98-fusions, NPM1 mutations, CDX deregulation, and MOZ-fusions lead to high level HOXA9 expression in acute leukemias. The mechanisms resulting in HOXA9 over expression are beginning to be defined and represent attractive therapeutic targets. Small molecules targeting MLL-fusion protein complex members, such as DOT1L and menin, have shown promising results in animal models, and a DOT1L inhibitor is currently being tested in clinical trials. Essential HOXA9 cofactors and collaborators are also being identified, including transcription factors PU.1 and C/EBPα, which are required for HOXA9-driven leukemia. HOXA9 targets including IGF1, CDX4, INK4A/INK4B/ARF, mir-21 and mir-196b and many others provide another avenue for potential drug development. Summary HOXA9 deregulation underlies a large subset of aggressive acute leukemias. Understanding the mechanisms regulating the expression and activity of HOXA9, along with its critical downstream targets, shows promise for the development of more selective and effective leukemia therapies.

Significance Acute myeloid leukemia (AML) is a highly heterogeneous form of cancer that results from the uncontrolled proliferation of primitive immune cells. Homeobox A9 (HOXA9) is an evolutionarily conserved transcription factor that is overexpressed in a large percentage of AML cases and is associated with a poor prognosis. Here, we show that CCAAT/enhancer binding protein alpha (C/EBPα), a transcription factor involved in immune cell development that is commonly mutated in AML, is a critical collaborator required for HOXA9-mediated leukemic transformation. We also establish that the cell cycle regulator cyclin-dependent kinase inhibitors Cdkn2a/b are corepressed by the Hoxa9–C/EBPα complex. These findings suggest a novel functional interaction between two leukemic transcription factors, HOXA9 and C/EBPα, that is altered in a large percentage of AML cases.