Unifying targeted therapy for leukemia in the era of PARP inhibition.

PARP inhibitors (PARPi) represent a novel class of targeted therapies that have conventionally been used for the treatment of BRCA1/2-mutated solid tumors. PARP1 being an indispensable component of the DNA repair machinery is essential for maintaining genomic integrity. Germline mutations or expression changes in genes compromising homologous recombination (HR)-mediated repair increases dependency on PARP1 and sensitizes these cells to PARP inhibition. Unlike solid tumors, hematologic malignancies do not frequently harbor BRCA1/2 mutations. PARP inhibition as a therapeutic strategy in blood disorders, therefore, did not receive the same importance. However, underlying epigenetic plasticity and leveraging transcriptional dependencies across molecular subtypes of leukemia has invigorated PARP inhibition-guided synthetic lethality in hematologic malignancies. For example, recent studies showing the importance of robust DNA repair machinery in acute myeloid leukemia (AML) increased the evidence of genomic instability associated with leukemia-driven mutations, and compromised repair pathways in certain subgroups of AML has shifted the focus on exploiting PARPi synthetic lethality in leukemia. Single-agent PARPi as well as combination with other targeted therapies has shown promising results in clinical trials involving patients with AML and myelodysplasia. In this study, we evaluated antileukemic potential of PARPi, understood the subtype-dependent differential responses, discussed recent clinical trials, and provided an outlook for future combination therapy strategies. Extensive genetic and epigenetic characterization, utilizing results from completed and ongoing studies will further help to determine specific subset of patients who may respond, and to establish PARPi as a mainstay in leukemia treatment.

KDM6 demethylases integrate DNA repair gene regulation and loss of KDM6A sensitizes human acute myeloid leukemia to PARP and BCL2 inhibition.

Acute myeloid leukemia (AML) is a heterogeneous, aggressive malignancy with dismal prognosis and with limited availability of targeted therapies. Epigenetic deregulation contributes to AML pathogenesis. KDM6 proteins are histone-3-lysine-27-demethylases that play context-dependent roles in AML. We inform that KDM6-demethylase function critically regulates DNA-damage-repair-(DDR) gene expression in AML. Mechanistically, KDM6 expression is regulated by genotoxic stress, with deficiency of KDM6A-(UTX) and KDM6B-(JMJD3) impairing DDR transcriptional activation and compromising repair potential. Acquired KDM6A loss-of-function mutations are implicated in chemoresistance, although a significant percentage of relapsed-AML has upregulated KDM6A. Olaparib treatment reduced engraftment of KDM6A-mutant-AML-patient-derived xenografts, highlighting synthetic lethality using Poly-(ADP-ribose)-polymerase-(PARP)-inhibition. Crucially, a higher KDM6A expression is correlated with venetoclax tolerance. Loss of KDM6A increased mitochondrial activity, BCL2 expression, and sensitized AML cells to venetoclax. Additionally, BCL2A1 associates with venetoclax resistance, and KDM6A loss was accompanied with a downregulated BCL2A1. Corroborating these results, dual targeting of PARP and BCL2 was superior to PARP or BCL2 inhibitor monotherapy in inducing AML apoptosis, and primary AML cells carrying KDM6A-domain mutations were even more sensitive to the combination. Together, our study illustrates a mechanistic rationale in support of a novel combination therapy for AML based on subtype-heterogeneity, and establishes KDM6A as a molecular regulator for determining therapeutic efficacy.

Emerging trends in chromatin remodeler plasticity in mesenchymal stromal cell function.

Emerging evidences highlight importance of epigenetic regulation and their integration with transcriptional and cell signaling machinery in determining tissue resident adult pluripotent mesenchymal stem/stromal cell (MSC) activity, lineage commitment, and multicellular development. Histone modifying enzymes and large multi-subunit chromatin remodeling complexes and their cell type-specific plasticity remain the central defining features of gene regulation and establishment of tissue identity. Modulation of transcription factor expression gradient ex vivo and concomitant flexibility of higher order chromatin architecture in response to signaling cues are exciting approaches to regulate MSC activity and tissue rejuvenation. Being an important constituent of the adult bone marrow microenvironment/niche, pathophysiological perturbation in MSC homeostasis also causes impaired hematopoietic stem/progenitor cell function in a non-cell autonomous mechanism. In addition, pluripotent MSCs can function as immune regulatory cells, and they reside at the crossroad of innate and adaptive immune response pathways. Research in the past few years suggest that MSCs/stromal fibroblasts significantly contribute to the establishment of immunosuppressive microenvironment in shaping antitumor immunity. Therefore, it is important to understand mesenchymal stromal epigenome and transcriptional regulation to leverage its applications in regenerative medicine, epigenetic memory-guided trained immunity, immune-metabolic rewiring, and precision immune reprogramming. In this review, we highlight the latest developments and prospects in chromatin biology in determining MSC function in the context of lineage commitment and immunomodulation.

Establishment of a Long-Term Co-culture Assay for Mesenchymal Stromal Cells and Hematopoietic Stem/Progenitors.

We describe a protocol for a long-term co-culture assay to study the contribution of mesenchymal stromal cells (MSCs) in regulating hematopoietic stem/progenitor cell (HSPC) activity. In addition, we describe the use of a clonogenic assay to determine myelo-erythroid differentiation. This long-term culture-initiating cell assay can be used for qualitative analysis of MSCs capable of supporting hematopoiesis and may also be used as a proxy readout to study HSPC repopulation. For complete details on the use and execution of this protocol, please refer to Sinha et al. (2020).

Evolving insights on histone methylome regulation in human acute myeloid leukemia pathogenesis and targeted therapy.

Acute myeloid leukemia (AML) is an aggressive, disseminated hematological malignancy associated with clonal selection of aberrant self-renewing hematopoietic stem cells and progenitors and poorly differentiated myeloid blasts. The most prevalent form of leukemia in adults, AML is predominantly an age-related disorder and accounts for more than 10,000 deaths per year in the United States alone. In comparison to solid tumors, AML has an overall low mutational burden, albeit more than 70% of AML patients harbor somatic mutations in genes encoding epigenetic modifiers and chromatin regulators. In the past decade, discoveries highlighting the role of DNA and histone modifications in determining cellular plasticity and lineage commitment have attested to the importance of epigenetic contributions to tumor cell de-differentiation and heterogeneity, tumor initiation, maintenance, and relapse. Orchestration in histone methylation levels regulates pluripotency and multicellular development. The increasing number of reversible methylation regulators being identified, including histone methylation writer, reader, and eraser enzymes, and their implications in AML pathogenesis have widened the scope of epigenetic reprogramming, with multiple drugs currently in various stages of preclinical and clinical trials. AML methylome also determines response to conventional chemotherapy, as well as AML cell interaction within a tumor-immune microenvironment ecosystem. Here we summarize the latest developments focusing on molecular derangements in histone methyltransferases (HMTs) and histone demethylases (HDMs) in AML pathogenesis. AML-associated HMTs and HDMs, through intricate crosstalk mechanisms, maintain an altered histone methylation code conducive to disease progression. We further discuss their importance in governing response to therapy, which can be used as a biomarker for treatment efficacy. Finally we deliberate on the therapeutic potential of targeting aberrant histone methylome in AML, examine available small molecule inhibitors in combination with immunomodulating therapeutic approaches and caveats, and discuss how future studies can enable posited epigenome-based targeted therapy to become a mainstay for AML treatment.

Pbrm1 Steers Mesenchymal Stromal Cell Osteolineage Differentiation by Integrating PBAF-Dependent Chromatin Remodeling and BMP/TGF-ß Signaling.

Bone morphogenic protein (BMP)/transforming growth factor ß (TGF-ß) signaling determines mesenchymal-stromal-cell (MSC) osteolineage commitment and tissue identity. However, molecular integration of developmental signaling with MSC-intrinsic chromatin regulation remains incompletely understood. SWI/SNF-(BAF) is an ATP-dependent chromatin remodeler implicated in multi-cellular development. We show that BMPs and long-term osteogenic signals in MSCs selectively induce expression of polybromo BAF (PBAF) components Pbrm1, Arid2, and Brd7. Loss of Pbrm1/Arid2/Brd7 profoundly impairs osteolineage gene expression and osteogenesis without compromising adipogenesis. Pbrm1 loss attenuates MSC in vivo ossification. Mechanistically, Pbrm1/PBAF deficiency impairs Smad1/5/8 activation through locus-specific epi-genomic remodeling, involving Pbrm1 bromodomains, along with transcriptional downregulation of Bmpr/TgfßrII affecting BMP-early-responsive gene expression. Gain of function of BmprIß, TgfßrII in PBAF-deficient MSCs partly restores Smad1/5/8 activation and osteogenesis. Pbrm1 loss further affects hematopoietic stem and progenitor activity through non-cell-autonomous regulation of microenvironment and niche-factor expression. Together, these findings reveal a link illustrating epi-genomic feedforward control of BMP/TGF-ß signaling to transcriptional and cellular plasticity in the mesenchymal microenvironment and account for stromal-SWI/SNF in hematopoiesis.

Transcriptomic Analysis Identifies RNA Binding Proteins as Putative Regulators of Myelopoiesis and Leukemia.

Acute myeloid leukemia (AML) is a common and aggressive hematological malignancy. Acquisition of heterogeneous genetic aberrations and epigenetic dysregulation lead to the transformation of hematopoietic stem cells (HSC) into leukemic stem cells (LSC), which subsequently gives rise to immature blast cells and a leukemic phenotype. LSCs are responsible for disease relapse as current chemotherapeutic regimens are not able to completely eradicate these cellular sub-populations. Therefore, it is critical to improve upon the existing knowledge of LSC specific markers, which would allow for specific targeting of these cells more effectively allowing for their sustained eradication from the cellular milieu. Although significant milestones in decoding the aberrant transcriptional network of various cancers, including leukemia, have been achieved, studies on the involvement of post-transcriptional gene regulation (PTGR) in disease progression are beginning to unfold. RNA binding proteins (RBPs) are key players in mediating PTGR and they regulate the intracellular fate of individual transcripts, from their biogenesis to RNA metabolism, via interactions with RNA binding domains (RBDs). In this study, we have used an integrative approach to systematically profile RBP expression and identify key regulatory RBPs involved in normal myeloid development and AML. We have analyzed RNA-seq datasets (GSE74246) of HSCs, common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), monocytes, LSCs, and blasts. We observed that normal and leukemic cells can be distinguished on the basis of RBP expression, which is indicative of their ability to define cellular identity, similar to transcription factors. We identified that distinctly co-expressing modules of RBPs and their subclasses were enriched in hematopoietic stem/progenitor (HSPCs) and differentiated monocytes. We detected expression of DZIP3, an E3 ubiquitin ligase, in HSPCs, knockdown of which promotes monocytic differentiation in cell line model. We identified co-expression modules of RBP genes in LSCs and among these, distinct modules of RBP genes with high and low expression. The expression of several AML-specific RBPs were also validated by quantitative polymerase chain reaction. Network analysis identified densely connected hubs of ribosomal RBP genes (rRBPs) with low expression in LSCs, suggesting the dependency of LSCs on altered ribosome dynamics. In conclusion, our systematic analysis elucidates the RBP transcriptomic landscape in normal and malignant myelopoiesis, and highlights the functional consequences that may result from perturbation of RBP gene expression in these cellular landscapes.

MBD3/NuRD loss participates with KDM6A program to promote DOCK5/8 expression and Rac GTPase activation in human acute myeloid leukemia.

Cancer genome sequencing studies have focused on identifying oncogenic mutations. However, mutational profiling alone may not always help dissect underlying epigenetic dependencies in tumorigenesis. Nucleosome remodeling and deacetylase (NuRD) is an ATP-dependent chromatin remodeling complex that regulates transcriptional architecture and is involved in cell fate commitment. We demonstrate that loss of MBD3, an important NuRD scaffold, in human primary acute myeloid leukemia (AML) cells associates with leukemic NuRD. Interestingly, CHD4, an intact ATPase subunit of leukemic NuRD, coimmunoprecipitates and participates with H3K27Me3/2-demethylase KDM6A to induce expression of atypical guanine nucleotide exchange factors, dedicator of cytokinesis (DOCK) 5 and 8 (DOCK5/8), promoting Rac GTPase signaling. Mechanistically, MBD3 deficiency caused loss of histone deacytelase 1 occupancy with a corresponding increase in KDM6A, CBP, and H3K27Ac on DOCK5/8 loci, leading to derepression of gene expression. Importantly, the Cancer Genome Atlas AML cohort reveals that DOCK5/ 8 levels are correlated with MBD3 and KDM6A, and DOCK5/ 8 expression is significantly increased in patients who are MBD3 low and KDM6A high with a poor survival. In addition, pharmacological inhibition of DOCK signaling selectively attenuates AML cell survival. Because MBD3 and KDM6A have been implicated in metastasis, our results may suggest a general phenomenon in tumorigenesis. Collectively, these findings provide evidence for MBD3-deficient NuRD in leukemia pathobiology and inform a novel epistasis between NuRD and KDM6A toward maintenance of oncogenic gene expression in AML.-Biswas, M., Chatterjee, S. S., Boila, L. D., Chakraborty, S., Banerjee, D., Sengupta, A. MBD3/NuRD loss participates with KDM6A program to promote DOCK5/8 expression and Rac GTPase activation in human acute myeloid leukemia.

A Potent Conformation-Constrained Synthetic Peptide Mimic of a Homeodomain Selectively Regulates Target Genes in Cells.

DNA, as a target for therapeutic intervention, remains largely unexplored. DLX-4, a homeodomain containing transcription factor, and its spliced isoforms play crucial roles in many aspects of cellular biochemistry and important roles in many diseases. A smaller peptide mimicking the homeodomain of the transcription factor DLX-4 was designed and synthesized by suitable conjoining of its modified DNA-binding elements. The peptide binds to DLX-4 target sites on the regulatory region of the globin gene cluster with native-like affinity and specificity in vitro. When conjugated to cell penetrating and nuclear localization sequences, it upregulated some of the genes repressed by DLX-4 or its isoforms, such as ß- and γ-globin genes in erythropoietin-induced differentiating CD34+ human hematopoietic stem/progenitor cells with high specificity by competing with the respective binding sites. Engineered peptides mimicking DNA-binding domains of transcription factors offer the potential for creating synthetic molecules for directly targeting DNA sites with high specificity.

SWI/SNF subunit expression heterogeneity in human aplastic anemia stem/progenitors.

Acquired aplastic anemia (AA) is a bone marrow (BM) failure associated with autoimmune destruction of hematopoietic stem cells (HSCs). Although somatic mutations have been identified in AA patients, mutations alone do not explain AA pathophysiology. SWI/SNF is an evolutionarily conserved, multi-subunit, ATP-dependent chromatin-remodeling protein complex that plays an important role in mammalian hematopoiesis. Herein, gene expression analysis identified a significant loss of the SWI/SNF core component SMARCC1, along with ARID1B, ACTL6A, and SMARCD1, in human AA BM CD34+ HSCs and hematopoietic stem and progenitor cells (HSPCs) compared with normal HSPCs. However, expression of SMARCA4, SMARCB1, SMARCD3, and DPF2 remained intact in our AA cohort. PBRM1, BRD7, and SMARCA2 expression were significantly upregulated in both untreated and follow-up AA patients. Clonal hematopoiesis in AA is associated with evolution to late clonal disorders, including myelodysplastic syndromes (MDS). Apart from SMARCD1 loss, we did not observe significant alteration of SWI/SNF expression in MDS HSPCs, indicating SWI/SNF differential expression in AA and MDS. In addition, except for ACTL6A, SWI/SNF expression was unaltered in aged HSPCs. Importantly, our results provide evidence for loss of SWI/SNF in AA, and may implicate AA HSPC-autonomous defective SWI/SNF regulation as an integral component of BM failure, in addition to autoimmune destruction of AA HSCs. These findings illustrate for the first time SWI/SNF subunit expression heterogeneity in human AA HSPCs and require prognostic validation in a larger cohort.

SMARCB1 Deficiency Integrates Epigenetic Signals to Oncogenic Gene Expression Program Maintenance in Human Acute Myeloid Leukemia.

SWI/SNF is an evolutionarily conserved multi-subunit chromatin remodeling complex that regulates epigenetic architecture and cellular identity. Although SWI/SNF genes are altered in approximately 25% of human malignancies, evidences showing their involvement in tumor cell-autonomous chromatin regulation and transcriptional plasticity are limiting. This study demonstrates that human primary acute myeloid leukemia (AML) cells exhibit near complete loss of SMARCB1 (BAF47 or SNF5/INI1) and SMARCD2 (BAF60B) associated with nucleation of SWI/SNFΔ SMARCC1 (BAF155), an intact core component of SWI/SNFΔ, colocalized with H3K27Ac to target oncogenic loci in primary AML cells. Interestingly, gene ontology (GO) term and pathway analysis suggested that SMARCC1 occupancy was enriched on genes regulating Rac GTPase activation, cell trafficking, and AML-associated transcriptional dysregulation. Transcriptome profiling revealed that expression of these genes is upregulated in primary AML blasts, and loss-of-function studies confirmed transcriptional regulation of Rac GTPase guanine nucleotide exchange factors (GEF) by SMARCB1. Mechanistically, loss of SMARCB1 increased recruitment of SWI/SNFΔ and associated histone acetyltransferases (HAT) to target loci, thereby promoting H3K27Ac and gene expression. Together, SMARCB1 deficiency induced GEFs for Rac GTPase activation and augmented AML cell migration and survival. Collectively, these findings highlight tumor suppressor role of SMARCB1 and illustrate SWI/SNFΔ function in maintaining an oncogenic gene expression program in AML.Implications: Loss of SMARCB1 in AML associates with SWI/SNFΔ nucleation, which in turn promotes Rac GTPase GEF expression, Rac activation, migration, and survival of AML cells, highlighting SWI/SNFΔ downstream signaling as important molecular regulator in AML. Mol Cancer Res; 16(5); 791-804. ©2018 AACR.

KDM6 and KDM4 histone lysine demethylases emerge as molecular therapeutic targets in human acute myeloid leukemia.

Acute myeloid leukemia (AML) remains an aggressive hematopoietic malignancy that is caused by proliferation of immature myeloid cells and is frequently characterized by perturbations in chromatin-modifying enzymes. Emerging evidence indicates that histone demethylases play a role in tumorigenesis. However, due to the complexity of this enormous family of histone-modifying enzymes, substrate redundancy, and context-specific roles, the contribution of each member remains ambiguous and targeting them remains challenging. Here, we analyzed expression of histone-3-lysine (H3K) demethylases and their cognate substrates in a cohort of de novo AML patients, which demonstrated that the expression of H3K27Me3/2-demethylases and selected members of H3K9Me3/2/1-demethylases are significantly increased in AML. KDM6 upregulation is associated with a global decrease in H3K27Me3 level. Importantly, our data show that pharmacological inhibition of H3K27Me3/2-demethylases or H3K9Me3/2-demethylases, either alone or in combination, could be considered an interesting molecular therapeutic modality in human AML independent of its subtype.

p62 is required for stem cell/progenitor retention through inhibition of IKK/NF-κB/Ccl4 signaling at the bone marrow macrophage-osteoblast niche.

In the bone marrow (BM), hematopoietic progenitors (HPs) reside in specific anatomical niches near osteoblasts (Obs), macrophages (MΦs), and other cells forming the BM microenvironment. A connection between immunosurveillance and traffic of HP has been demonstrated, but the regulatory signals that instruct the immune regulation of HP circulation are unknown. We discovered that the BM microenvironment deficiency of p62, an autophagy regulator and signal organizer, results in loss of autophagic repression of macrophage contact-dependent activation of Ob NF-κB signaling. Consequently, Ob p62-deficient mice lose bone, Ob Ccl4 expression, and HP chemotaxis toward Cxcl12, resulting in egress of short-term hematopoietic stem cells and myeloid progenitors. Finally, Ccl4 expression and myeloid progenitor egress are reversed by deficiency of the p62 PB1-binding partner Nbr1. A functional "MΦ-Ob niche" is required for myeloid progenitor/short-term stem cell retention, in which Ob p62 is required to maintain NF-κB signaling repression, osteogenesis, and BM progenitor retention.

Malignant peripheral nerve sheath tumour presenting with Horner's syndrome.

A young male presented with clinical and radiological features of right apical lung mass and Horner's syndrome. Subsequently the patient was diagnosed as a case of malignant peripheral nerve sheath tumour (MPNST) at the apex of right lung originating from an intercostal nerve and compressing ipsilateral cervical sympathetic plexus and lower cord of brachial plexus, in a case of neurofibromatosis type 1.

Vav3 collaborates with p190-BCR-ABL in lymphoid progenitor leukemogenesis, proliferation, and survival.

Despite the introduction of tyrosine kinase inhibitor therapy, the prognosis for p190-BCR-ABL(+) acute lymphoblastic leukemia remains poor. In the present study, we present the cellular and molecular roles of the Rho GTPase guanine nucleotide exchange factor Vav in lymphoid leukemogenesis and explore the roles of Vav proteins in BCR-ABL-dependent signaling. We show that genetic deficiency of the guanine nucleotide exchange factor Vav3 delays leukemogenesis by p190-BCR-ABL and phenocopies the effect of Rac2 deficiency, a downstream effector of Vav3. Compensatory up-regulation of expression and activation of Vav3 in Vav1/Vav2-deficient B-cell progenitors increases the transformation ability of p190-BCR-ABL. Vav3 deficiency induces apoptosis of murine and human leukemic lymphoid progenitors, decreases the activation of Rho GTPase family members and p21-activated kinase, and is associated with increased Bad phosphorylation and up-regulation of Bax, Bak, and Bik. Finally, Vav3 activation only partly depends on ABL TK activity, and Vav3 deficiency collaborates with tyrosine kinase inhibitors to inhibit CrkL activation and impair leukemogenesis in vitro and in vivo. We conclude that Vav3 represents a novel specific molecular leukemic effector for multitarget therapy in p190-BCR-ABL-expressing acute lymphoblastic leukemia.

Connexin-43 in the osteogenic BM niche regulates its cellular composition and the bidirectional traffic of hematopoietic stem cells and progenitors.

Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an in vivo model of constitutive deficiency of Cx43 in OB/P. The deficiency of Cx43 in OB/P cells does not impair the steady state hematopoiesis, but disrupts the directional trafficking of HSC/progenitors (Ps) between the bone marrow (BM) and peripheral blood (PB). OB/P Cx43 is a crucial positive regulator of transstromal migration and homing of both HSCs and progenitors in an irradiated microenvironment. However, OB/P Cx43 deficiency in nonmyeloablated animals does not result in a homing defect but induces increased endosteal lodging and decreased mobilization of HSC/Ps associated with proliferation and expansion of Cxcl12-secreting mesenchymal/osteolineage cells in the BM HM in vivo. Cx43 controls the cellular content of the BM osteogenic microenvironment and is required for homing of HSC/Ps in myeloablated animals.

Squamous cell carcinoma of lung metastasizinig in breast.

Metastasis in breast from extra-mammary malignancy is rare and lung is the third most common primary site when such a metastasis occurs. Small cell carcinoma and adenocarcinoma are two histological varieties of lung carcinoma that may metastasize to breast and squamous cell type is very rare. Here we report a case of squamous cell carcinoma of lung that metastasized in the breast and mimicked primary breast carcinoma.

Bmi1 reprograms CML B-lymphoid progenitors to become B-ALL-initiating cells.

The characterization and targeting of Philadelphia chromosome positive (Ph(+)) acute lymphoblastic leukemia (ALL)-initiating cells remains unresolved. Expression of the polycomb protein Bmi1 is up-regulated in patients with advanced stages of chronic myelogenous leukemia (CML). We report that Bmi1 transforms and reprograms CML B-lymphoid progenitors into stem cell leukemia (Scl) promoter-driven, self-renewing, leukemia-initiating cells to result in B-lymphoid leukemia (B-ALL) in vivo. In vitro, highly proliferating and serially replatable myeloid and lymphoid colony-forming cultures could be established from BCR-ABL and Bmi1 coexpressing progenitors. However, unlike in vivo expanded CML B-lymphoid progenitors, hematopoietic stem cells, or multipotent progenitors, coexpressing BCR-ABL and Bmi1 did not initiate or propagate leukemia in a limiting dilution assay. Inducible genetic attenuation of BCR-ABL reversed Bmi1-driven B-ALL development, which was accompanied by induction of apoptosis of leukemic B-lymphoid progenitors and by long-term animal survival, suggesting that BCR-ABL is required to maintain B-ALL and that BCR-ABL and Bmi1 cooperate toward blast transformation in vivo. Our data indicate that BCR-ABL targeting itself is required to eradicate Ph(+)/Bmi1(+) B-ALL-initiating cells and confirm their addiction to BCR-ABL signaling.

Atypical protein kinase C (aPKCzeta and aPKClambda) is dispensable for mammalian hematopoietic stem cell activity and blood formation.

The stem-cell pool is considered to be maintained by a balance between symmetric and asymmetric division of stem cells. The cell polarity model proposes that the facultative use of symmetric and asymmetric cell division is orchestrated by a polarity complex consisting of partitioning-defective proteins Par3 and Par6, and atypical protein kinase C (aPKCζ and aPKCλ), which regulates planar symmetry of dividing stem cells with respect to the signaling microenvironment. However, the role of the polarity complex is unexplored in mammalian adult stem-cell functions. Here we report that, in contrast to accepted paradigms, polarization and activity of adult hematopoietic stem cell (HSC) do not depend on either aPKCζ or aPKCλ or both in vivo. Mice, having constitutive and hematopoietic-specific (Vav1-Cre) deletion of aPKCζ and aPKCλ, respectively, have normal hematopoiesis, including normal HSC self-renewal, engraftment, differentiation, and interaction with the bone marrow microenvironment. Furthermore, inducible complete deletion of aPKCλ (Mx1-Cre) in aPKCζ(-/-) HSC does not affect HSC polarization, self-renewal, engraftment, or lineage repopulation. In addition, aPKCζ- and aPKCλ-deficient HSCs elicited a normal pattern of hematopoietic recovery secondary to myeloablative stress. Taken together, the expression of aPKCζ, aPKCλ, or both are dispensable for primitive and adult HSC fate determination in steady-state and stress hematopoiesis, contrary to the hypothesis of a unique, evolutionary conserved aPKCζ/λ-directed cell polarity signaling mechanism in mammalian HSC fate determination.