Supplementary MaterialsSupplementary Information 41467_2018_7846_MOESM1_ESM

Supplementary MaterialsSupplementary Information 41467_2018_7846_MOESM1_ESM. atypical protein kinase C (aPKC). While the isoform aPKC behaves as a leukemic suppressor, aPKC/ is critically required for oncogenic progenitor proliferation, survival, and B-cell differentiation arrest, but not for normal B-cell lineage differentiation. In vitro and in vivo B-cell transformation by BCR-ABL requires the downregulation of key genes in the B-cell differentiation program through an aPKC /-Erk dependent Etv5/Satb2 chromatin repressive signaling complex. Genetic or pharmacological targeting of aPKC impairs human oncogenic addicted leukemias. Therefore, the aPKC/-SATB2 signaling cascade is required for leukemic BCR-ABL+ B-cell progenitor transformation and is amenable to non-tyrosine kinase inhibition. Introduction B lymphoid leukemia arises from hematopoietic stem cells (HSC) or B-cell progenitors, so-called leukemic progenitors that have acquired a transforming, leukemia-initiating event. A major example of a leukemia-initiating event is the expression of p210-BCR-ABL, which is?the product of t(9;22)(q34;q11) translocation, and?is necessary and sufficient for the development and Staurosporine progression of chronic myelogenous leukemia (CML)1. The transforming ability of Staurosporine BCR-ABL is dependent on its deregulated tyrosine kinase (TK) activity leading to its auto-phosphorylation, recruitment of adaptor proteins, and subsequent activation of downstream signaling pathways, including Ras, extracellular-signal-regulated kinase (ERK), Akt, c-Jun activated kinase (JNK), p38, CrkL, signal transducer and activator of transcription 5 (STAT5), and nuclear factor-B (NF-kB)2. Progression of BCR-ABL+ leukemia from the chronic phase to the poor prognosis blast crisis phase is accompanied by increased BCR-ABL expression, genetic instability, increased proliferation, reduced apoptosis, and a blockade of differentiation where myeloid or lymphoid progenitors/precursors fail to differentiate, resulting in the development of acute myelogenous leukemia (AML) or B-cell acute lymphoblastic leukemia Staurosporine (B-ALL)2C5. Genetic abnormalities such as increased Myc expression6, upregulation Fzd4 of Bmi17, homozygous deletion of exon 2 of diet to induce BCR-ABL expression. WT and aPKC?/? secondary chimeric mice developed B-ALL with median survival of 61.5 days and 52.5 days, respectively (Fig.?2a). aPKC?/? chimeric mice died significantly earlier than the WT group (and was upregulated in DKO group, with no significant changes in other PKC isoforms (Supplemental Table?1). The upregulation of mRNA expression did not translate into increased protein levels. However, PKC level is increased in aPKC/ and DKO cells and decreased in aPKC?/? progenitors, which is consistent with a possible tumor suppressor role of PKC (Supplementary Figure?3D). Open in a separate window Fig. 3 aPKC deficiency impairs proliferation, survival and B cell differentiation arrest. a Comparative transcriptome and gene-ontology (GO) pathway analyses of the differential expression of genes in WT and DKO leukemic B-cell progenitors showing the differential regulation of genes involved in proliferation, cell cycle regulation, B cell differentiation network, and histone and chromatin modifications. Pathways shown?in Blue?-?proliferation and cell cycle regulation; in Red?-?B cell differentiation network; in Green?-?histone and chromatin modification. b In vivo BrDU uptake by leukemic B-cell progenitors in Scl/P210; WT, aPKC?/?, aPKC/, and DKO chimeric mice. c FACS quantification of annexin V-binding of WT, aPKC?/?, aPKC/ and DKO leukemic B-cell progenitors. d Representative example of western blots of p-ERK1/2, ERK1/2, p-MEK1/2, MEK1/2, aPKC, and Actin in WT, aPKC?/?, aPKC/, and DKO leukemic B-cell progenitors. Activation of MEK/ERK MAPK Staurosporine pathway is impaired in aPKC deficient leukemic B-cell progenitors. Western blots with different exposure time for phospho-Erk1/2 and phospho-Mek1/2 were presented to show minimal expression; Low (15?sec), high (1?min). e Representative example of the analyses of Rac GTPase activation by specific effector pulldown (PAK-PBD agarose) assay in leukemic B-cell progenitors derived from WT, aPKC?/?, aPKC?/? and DKO chimeric mice. f FACS-quantification of proB, preB and immature/mature B cells in the BM of WT, aPKC?/?, aPKC/ and DKO chimeric mice. Data are presented as mean??SD of a minimum of three independent experiments. *(cyclin-D1), and increased (p21), (p27) levels in aPKC?/? or DKO cells (Supplementary Figure?3E). Furthermore, aPKC?/? and DKO leukemic B-cell progenitors showed increased apoptosis (Fig.?3c). In contrast, deletion of aPKC alone significantly increased the proliferation and survival of leukemic B-cell progenitors (Fig.?3b, c). To.