Delivery of PEGylated liposomal doxorubicin by bispecific antibodies improves treatment in models of high-risk childhood leukemia

High-risk childhood leukemia has a poor prognosis because of treatment failure and toxic side effects of therapy. Drug encapsulation into liposomal nanocarriers has shown clinical success at improving biodistribution and tolerability of chemotherapy. However, enhancements in drug efficacy have been limited because of a lack of selectivity of the liposomal formulations for the cancer cells. Here, we report on the generation of bispecific antibodies (BsAbs) with dual binding to a leukemic cell receptor, such as CD19, CD20, CD22, or CD38, and methoxy polyethylene glycol (PEG) for the targeted delivery of PEGylated liposomal drugs to leukemia cells. This liposome targeting system follows a "mix-and-match" principle where BsAbs were selected on the specific receptors expressed on leukemia cells. BsAbs improved the targeting and cytotoxic activity of a clinically approved and low-toxic PEGylated liposomal formulation of doxorubicin (Caelyx) toward leukemia cell lines and patient-derived samples that are immunophenotypically heterogeneous and representative of high-risk subtypes of childhood leukemia. BsAb-assisted improvements in leukemia cell targeting and cytotoxic potency of Caelyx correlated with receptor expression and were minimally detrimental in vitro and in vivo toward expansion and functionality of normal peripheral blood mononuclear cells and hematopoietic progenitors. Targeted delivery of Caelyx using BsAbs further enhanced leukemia suppression while reducing drug accumulation in the heart and kidneys and extended overall survival in patient-derived xenograft models of high-risk childhood leukemia. Our methodology using BsAbs therefore represents an attractive targeting platform to potentiate the therapeutic efficacy and safety of liposomal drugs for improved treatment of high-risk leukemia

Integrative Genomics Identifies the Molecular Basis of Resistance to Azacitidine Therapy in Myelodysplastic Syndromes

© 2017 The Author(s) Myelodysplastic syndromes and chronic myelomonocytic leukemia are blood disorders characterized by ineffective hematopoiesis and progressive marrow failure that can transform into acute leukemia. The DNA methyltransferase inhibitor 5-azacytidine (AZA) is the most effective pharmacological option, but only ∼50% of patients respond. A response only manifests after many months of treatment and is transient. The reasons underlying AZA resistance are unknown, and few alternatives exist for non-responders. Here, we show that AZA responders have more hematopoietic progenitor cells (HPCs) in the cell cycle. Non-responder HPC quiescence is mediated by integrin α5 (ITGA5) signaling and their hematopoietic potential improved by combining AZA with an ITGA5 inhibitor. AZA response is associated with the induction of an inflammatory response in HPCs in vivo. By molecular bar coding and tracking individual clones, we found that, although AZA alters the sub-clonal contribution to different lineages, founder clones are not eliminated and continue to drive hematopoiesis even in complete responders

Expansion of human hematopoietic stem/progenitor cells on decellularized matrix scaffolds

Umbilical cord blood (UCB) is one of the richest sources for hematopoietic stem/progenitor cells (HSPCs), with more than 3000 transplantations performed each year for the treatment of leukemia and other bone marrow, immunological, and hereditary diseases. However, transplantation of single cord blood units is mostly restricted to children, due to the limited number of HSPC per unit. This unit develops a method to increase the number of HSPCs in laboratory conditions by using cell-free matrices from bone marrow cells that mimic \u27human-body niche-like\u27 conditions as biological scaffolds to support the ex vivo expansion of HSPCs. In this unit, we describe protocols for the isolation and characterization of HSPCs from UCB and their serum-free expansion on decellularized matrices. This method may also help to provide understanding of the biochemical organization of hematopoietic niches and lead to suggestions regarding the design of tissue engineering-based biomimetic scaffolds for HSPC expansion for clinical applications

miR-10a as a therapeutic target and predictive biomarker for MDM2 inhibition in acute myeloid leukemia

Pharmacological inhibition of MDM2/4, which activates the critical tumor suppressor p53, has been gaining increasing interest as a strategy for the treatment of acute myeloid leukemia (AML). While clinical trials of MDM2 inhibitors have shown promise, responses have been confined to largely molecularly undefined patients, indicating that new biomarkers and optimized treatment strategies are needed. We previously reported that the microRNA miR-10a is strongly overexpressed in some AML, and demonstrate here that it modulates several key members of the p53/Rb network, including p53 regulator MDM4, Rb regulator RB1CC1, p21 regulator TFAP2C, and p53 itself. The expression of both miR-10a and its downstream targets were strongly predictive of MDM2 inhibitor sensitivity in cell lines, primary AML specimens, and correlated to response in patients treated with both MDM2 inhibitors and cytarabine. Furthermore, miR-10a inhibition induced synergy between MDM2 inhibitor Nutlin-3a and cytarabine in both in vitro and in vivo AML models. Mechanistically this synergism primarily occurs via the p53-mediated activation of cytotoxic apoptosis at the expense of cytoprotective autophagy. Together these findings demonstrate that miR-10a may be useful as both a biomarker to identify patients most likely to respond to cytarabine+MDM2 inhibition and also a druggable target to increase their efficacy

MAPK/ERK2 phosphorylates ERG at serine 283 in leukemic cells and promotes stem cell signatures and cell proliferation

© 2016 Macmillan Publishers Limited. Aberrant ERG (v-ets avian erythroblastosis virus E26 oncogene homolog) expression drives leukemic transformation in mice and high expression is associated with poor patient outcomes in acute myeloid leukemia (AML) and T-acute lymphoblastic leukemia (T-ALL). Protein phosphorylation regulates the activity of many ETS factors but little is known about ERG in leukemic cells. To characterize ERG phosphorylation in leukemic cells, we applied liquid chromatography coupled tandem mass spectrometry and identified five phosphorylated serines on endogenous ERG in T-ALL and AML cells. S283 was distinct as it was abundantly phosphorylated in leukemic cells but not in healthy hematopoietic stem and progenitor cells (HSPCs). Overexpression of a phosphoactive mutant (S283D) increased expansion and clonogenicity of primary HSPCs over and above wild-type ERG. Using a custom antibody, we screened a panel of primary leukemic xenografts and showed that ERG S283 phosphorylation was mediated by mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling and in turn regulated expression of components of this pathway. S283 phosphorylation facilitates ERG enrichment and transactivation at the ERG +85 HSPC enhancer that is active in AML and T-ALL with poor prognosis. Taken together, we have identified a specific post-translational modification in leukemic cells that promotes progenitor proliferation and is a potential target to modulate ERG-driven transcriptional programs in leukemia

Overexpression of ERG in cord blood progenitors promotes expansion and recapitulates molecular signatures of high ERG leukemias

© 2015 Macmillan Publishers Limited. High expression of the ETS family transcription factor ERG is associated with poor clinical outcome in acute myeloid leukemia (AML) and acute T-cell lymphoblastic leukemia (T-ALL). In murine models, high ERG expression induces both T-ALL and AML. However, no study to date has defined the effect of high ERG expression on primary human hematopoietic cells. In the present study, human CD34+ cells were transduced with retroviral vectors to elevate ERG gene expression to levels detected in high ERG AML. RNA sequencing was performed on purified populations of transduced cells to define the effects of high ERG on gene expression in human CD34+ cells. Integration of the genome-wide expression data with other data sets revealed that high ERG drives an expression signature that shares features of normal hematopoietic stem cells, high ERG AMLs, early T-cell precursor-ALLs and leukemic stem cell signatures associated with poor clinical outcome. Functional assays linked this gene expression profile to enhanced progenitor cell expansion. These results support a model whereby a stem cell gene expression network driven by high ERG in human cells enhances the expansion of the progenitor pool, providing opportunity for the acquisition and propagation of mutations and the development of leukemia

Induction of Muscle Regenerative Multipotent Stem Cells from Human Adipocytes by PDGF-AB and 5-Azacytidine

ABSTRACTTerminally differentiated murine osteocytes and adipocytes can be reprogrammed using platelet-derived growth factor–AB and 5-Azacytidine into multipotent stem cells with stromal cell characteristics. To generate a product that is amenable for therapeutic application, we have modified and optimised culture conditions to reprogram human adipocytes into induced multipotent stem cells (iMS) and expand them in vitro. The basal transcriptomes of adipocyte-derived iMS cells and matched adipose-tissue-derived mesenchymal stem cells were remarkably similar. However, there were distinct changes in histone modifications and CpG methylation at cis-regulatory regions consistent with an epigenetic landscape that was primed for tissue development and differentiation. In a non-specific tissue injury xenograft model, iMS cells contributed directly to new muscle, bone, cartilage and blood vessels with no evidence of teratogenic potential. In a cardiotoxin muscle injury model, iMS cells contributed specifically to satellite cells and myofibres without ectopic tissue formation. Taken together, human adipocyte derived iMS cells regenerate tissues in a context dependent manner without ectopic or neoplastic growth.</jats:p

Induction of muscle-regenerative multipotent stem cells from human adipocytes by PDGF-AB and 5-azacytidine

Terminally differentiated murine osteocytes and adipocytes can be reprogrammed using platelet-derived growth factor-AB and 5-azacytidine into multipotent stem cells with stromal cell characteristics. We have now optimized culture conditions to reprogram human adipocytes into induced multipotent stem (iMS) cells and characterized their molecular and functional properties. Although the basal transcriptomes of adipocyte-derived iMS cells and adipose tissue-derived mesenchymal stem cells were similar, there were changes in histone modifications and CpG methylation at cis-regulatory regions consistent with an epigenetic landscape that was primed for tissue development and differentiation. In a non-specific tissue injury xenograft model, iMS cells contributed directly to muscle, bone, cartilage, and blood vessels, with no evidence of teratogenic potential. In a cardiotoxin muscle injury model, iMS cells contributed specifically to satellite cells and myofibers without ectopic tissue formation. Together, human adipocyte-derived iMS cells regenerate tissues in a context-dependent manner without ectopic or neoplastic growth

Aberrant expression of miR-9/9* in myeloid progenitors inhibits neutrophil differentiation by post-transcriptional regulation of ERG

Aberrant post-transcriptional regulation by microRNAs (miRNAs) has been shown to be involved in the pathogenesis of acute myeloid leukemia (AML). In a previous study, we performed a large functional screen using a retroviral barcoded miRNA expression library. Here, we report that overexpression of miR-9/9* in myeloid 32D cell line (32D-miR-9/9*) had profound impact on granulocyte colony-stimulating factor-induced differentiation. Further in vitro studies showed that enforced expression of miR-9/9* blocked normal neutrophil development in 32D and in primary murine lineage-negative bone marrow cells. We examined the expression of miR-9/9* in a cohort of 647 primary human AMLs. In most cases, miR-9 and miR-9* were significantly upregulated and their expression levels varied according to AML subtype, with the highest expression in MLL-related leukemias harboring 11q23 abnormalities and the lowest expression in AML cases with t(8;21) and biallelic mutations in CEBPA. Gene expression profiling of AMLs with high expression of miR-9/9* and 32D-miR-9/9* identified ETS-related gene (Erg) as the only common potential target. Upregulation of ERG in 32D cells rescued miR-9/9*-induced block in neutrophil differentiation. Taken together, this study demonstrates that miR-9/9* are aberrantly expressed in most of AML cases and interfere with normal neutrophil differentiation by downregulation of ERG