Abstract
The molecular mechanisms that control leukemic transformation in B-cell acute lymphoblastic leukemia remain unclear. Leukemic stem cells/progenitors (LSC/P) hijack common pathways utilized by hematopoietic stem cells/progenitors (HSC/P) while they also activate other unique pathways that result in increased self-renewal and differentiation arrest susceptible of therapeutic intervention. A major regulator of HSC self-renewal vs differentiation is the distribution of polarized cell fate determinants. Atypical protein kinase C (aPKC)-ζ and aPKCi in humans or its homologue aPKCλ in mice are the catalytic components of the apical polarity complex PAR3-PAR6. aPKCs have also been argued to function as tumor suppressors. We have previously shown that the genetic deficiency of aPKC activity is dispensable for normal HSC/P activity (Sengupta A et al., PNAS 2011). We had found that primary human and murine BCR-ABL+ LSC/P have ~2-3 fold increased activation of aPKCλ/i. To investigate the possible role of aPKCs in LSC/P activity, we utilized a lymphoid blast-crisis chronic myelogenous leukemia model and evaluated the role aPKCζ and aPKCλ inducible deficiency in leukemic progression. After leukemia induction resulting from doxcycycline withdrawal in Scl-tTA/TRE-BCR-ABL mice that were also transgenic for Mx1Cre, aPKCζ-/- and/or aPKCλflox/flox, leukemic mice (dox-off) were treated with polyI:C to induce deletion of aPKCλ. aPKCλΔ/Δ LSC (Lin-cKit+Sca1+CD135-CD34-) and progenitors (CFU-C) of transplanted recipient mice were 40 and 87% reduced, respectively, suggesting a critical role of aPKCλ in the maintenance of LSC/P. Upon serial transplantation of bone marrow (BM) from CML animals, all WT and aPKCζ-/- recipient mice died by 6-10 weeks of B-ALL. However, mice transplanted with aPKCλΔ/Δ or aPKCζ-/-;aPKCλΔ/Δ BM cells did not die after serial transplantation followed for over one year, despite maintaining leukemic B-cells in circulation and hematopoietic tissues with catalytically active BCR-ABL expression. Leukemic ProB/PreB cells deficient in aPKCλ activity showed ~67% reduced proliferation and 2-fold higher apoptosis than WT and aPKCζ deficient ProB/PreB cells while non-leukemic aPKCλ deficient B-cell lymphopoiesis was grossly normal. Similarly, shRNA-mediated knock-down of aPKCi, in CML patient derived LSC/P led to reduced proliferation, increased apoptosis and enhanced differentiation indicating that aPKCi is also required for the survival and maintenance of human LSC/P. Strikingly, the B-cell differentiation program was restored in aPKCλΔ/Δ mice with expansion of IgM expressing B cells. Downstream, the activation of CrkL, MEK/ERK and Myc signaling pathways were attenuated in absence of aPKCλ in leukemic B-progenitor cells. Unlike non-leukemic B-cell progenitors, the transcriptome of aPKCλ-deficient leukemic B-progenitors showed up-regulation of the B-lymphoid differentiation gene network (Pax5, Ebf1, Ikzf1, Ikzf2, Ikzf3, Rag1 & Rag2). Interestingly, Myc protein expression was reduced and the cell fate determinant Numb mislocalized in aPKCλΔ/Δ leukemic B-cell progenitors, with predominant nuclear distribution. The mRNA/protein expression of Satb2, a chromatin modifier controlling Myc and Numb expression as well as B-cell differentiation, was significantly reduced in aPKCλ-deficient leukemic B-cell progenitors while the expression of its antagonist Satb1 was unchanged. Chromatin immunoprecipitation analysis of leukemic B-cell progenitors showed ~16-200 fold increased enrichment of Satb2 binding to promoter sequences of Cdkn2a and B-cell differentiation genes Pax5 and Ebf1 over non-leukemic B-cell progenitors. Finally, forced expression of Satb2 in aPKCλΔ/Δ deficient LSC/P rescued leukemic proliferation and B-cell differentiation arrest in vivo indicating that Satb2, downstream of aPKCλ, is required for BCR-ABL+ LSC/P activity. Taken together, these data indicate that aPKCλacts as an oncogene in leukemic B-cell progenitors. Loss of aPKCλ reduces LSC/proB proliferation and survival, and restores B-cell differentiation through combined reduction of MAPK activation, Numb mislocalization and differential expression/activity of the chromatin modifier Satb2. This data defines the role of aPKCλ in LSC/P activity and identifies aPKCλ as a new target for pharmacological intervention dispensable for normal lympho-hematopoiesis.
Disclosures
Cancelas: US Army Medical Research and Material Command (Award W81XWH-15-C-0047) / Department of Defense: Research Funding; Terumo BCT: Research Funding; Cellphire, Inc.: Membership on an entity's Board of Directors or advisory committees; Cerus Corporation: Research Funding; Citra Labs, Inc.: Research Funding; Leukemia & Lymphoma Society of North America: Research Funding; William & Lawrence Hughes Foundation: Research Funding; Haemonetics, Inc.: Research Funding; National Institutes of Health: Research Funding; New Health Sciences, Inc.: Membership on an entity's Board of Directors or advisory committees.
Assessing the role of the T-box transcription factor Eomes in B cell differentiation during either Th1 or Th2 cell-biased responses