Cell-cell interactome of the hematopoietic niche and its changes in acute myeloid leukemia.

The bone marrow (BM) is a complex microenvironment, coordinating the production of billions of blood cells every day. Despite its essential role and its relevance to hematopoietic diseases, this environment remains poorly characterized. Here we present a high-resolution characterization of the niche in health and acute myeloid leukemia (AML) by establishing a single-cell gene expression database of 339,381 BM cells. We found significant changes in cell type proportions and gene expression in AML, indicating that the entire niche is disrupted. We then predicted interactions between hematopoietic stem and progenitor cells (HSPCs) and other BM cell types, revealing a remarkable expansion of predicted interactions in AML that promote HSPC-cell adhesion, immunosuppression, and cytokine signaling. In particular, predicted interactions involving transforming growth factor ß1 (TGFB1) become widespread, and we show that this can drive AML cell quiescence in vitro. Our results highlight potential mechanisms of enhanced AML-HSPC competitiveness and a skewed microenvironment, fostering AML growth.

Concurrent transposon engineering and CRISPR/Cas9 genome editing of primary CLL-1 chimeric antigen receptor-natural killer cells.

BACKGROUND: Natural killer (NK) cell genome editing promises to enhance the innate and alloreactive anti-tumor potential of NK cell adoptive transfer. DNA transposons are versatile non-viral gene vectors now being adapted to primary NK cells, representing important tools for research and clinical product development. AIMS AND METHODS: We set out to generate donor-derived, primary chimeric antigen receptor (CAR)-NK cells by combining the TcBuster transposon system with Epstein-Barr virus-transformed lymphoblastoid feeder cell-mediated activation and expansion. RESULTS: This approach allowed for clinically relevant NK-cell expansion capability and CAR expression, which was further enhanced by immunomagnetic selection based on binding to the CAR target protein.The resulting CAR-NK cells targeting the myeloid associated antigen CLL-1 efficiently targeted CLL-1-positive AML cell lines and primary AML populations, including a population enriched for leukemia stem cells. Subsequently, concurrent delivery of CRISPR/Cas9 cargo was applied to knockout the NK cell cytokine checkpoint cytokine-inducible SH2-containing protein (CIS, product of the CISH gene), resulting in enhanced cytotoxicity and an altered NK cell phenotype. CONCLUSIONS: This report contributes a promising application of transposon engineering to donor-derived NK cells and emphasizes the importance of feeder mediated NK cell activation and expansion to current protocols.

Recreating the Bone Marrow Microenvironment to Model Leukemic Stem Cell Quiescence.

The main challenge in the treatment of acute myeloid leukemia (AML) is relapse, as it has no good treatment options and 90% of relapsed patients die as a result. It is now well accepted that relapse is due to a persisting subset of AML cells known as leukemia-initiating cells or leukemic stem cells (LSCs). Hematopoietic stem cells (HSCs) reside in the bone marrow microenvironment (BMM), a specialized niche that coordinates HSC self-renewal, proliferation, and differentiation. HSCs are divided into two types: long-term HSCs (LT-HSCs) and short-term HSCs, where LT-HSCs are typically quiescent and act as a reserve of HSCs. Like LT-HSCs, a quiescent population of LSCs also exist. Like LT-HSCs, quiescent LSCs have low metabolic activity and receive pro-survival signals from the BMM, making them resistant to drugs, and upon discontinuation of therapy, they can become activated and re-establish the disease. Several studies have shown that the activation of quiescent LSCs may sensitize them to cytotoxic drugs. However, it is very difficult to experimentally model the quiescence-inducing BMM. Here we report that culturing AML cells with bone marrow stromal cells, transforming growth factor beta-1 and hypoxia in a three-dimensional system can replicate the quiescence-driving BMM. A quiescent-like state of the AML cells was confirmed by reduced cell proliferation, increased percentage of cells in the G0 cell cycle phase and a decrease in absolute cell numbers, expression of markers of quiescence, and reduced metabolic activity. Furthermore, the culture could be established as co-axial microbeads, enabling high-throughput screening, which has been used to identify combination drug treatments that could break BMM-mediated LSC quiescence, enabling the eradication of quiescent LSCs.

Hematopoietic versus leukemic stem cell quiescence: Challenges and therapeutic opportunities.

Hematopoietic stem cells (HSC) are responsible for the production of mature blood cells. To ensure that the HSC pool does not get exhausted over the lifetime of an individual, most HSCs are in a state of quiescence with only a small proportion of HSCs dividing at any one time. HSC quiescence is carefully controlled by both intrinsic and extrinsic, niche-driven mechanisms. In acute myeloid leukemia (AML), the leukemic cells overtake the hematopoietic bone marrow niche where they acquire a quiescent state. These dormant AML cells are resistant to chemotherapeutics. Because they can re-establish the disease after therapy, they are often termed as quiescent leukemic stem cells (LSC) or leukemia-initiating cells. While advancements are being made to target particular driver mutations in AML, there is less focus on how to tackle the drug resistance of quiescent LSCs. This review summarises the current knowledge on the biochemical characteristics of quiescent HSCs and LSCs, the intracellular signaling pathways and the niche-driven mechanisms that control quiescence and the key differences between HSC- and LSC-quiescence that may be exploited for therapy.

Repression of Mcl-1 expression by the CDC7/CDK9 inhibitor PHA-767491 overcomes bone marrow stroma-mediated drug resistance in AML.

Acute myeloid leukaemia (AML) is an aggressive cancer with 50-75% of patients relapsing even after successful chemotherapy. The role of the bone marrow microenvironment (BMM) in protecting AML cells from chemotherapeutics and causing consequent relapse is increasingly recognised. However the role that the anti-apoptotic Bcl-2 proteins play as effectors of BMM-mediated drug resistance are less understood. Here we show that bone marrow mesenchymal stromal cells (BMSC) provide resistance to AML cells against BH3-mimetics, cytarabine and daunorubicin, but this is not mediated by Bcl-2 and/or Bcl-XL as previously thought. Instead, BMSCs induced Mcl-1 expression over Bcl-2 and/or Bcl-XL in AML cells and inhibition of Mcl-1 with a small-molecule inhibitor, A1210477, or repressing its expression with the CDC7/CDK9 dual-inhibitor, PHA-767491 restored sensitivity to BH3-mimetics. Furthermore, combined inhibition of Bcl-2/Bcl-XL and Mcl-1 could revert BMSC-mediated resistance against cytarabine + daunorubicin. Importantly, the CD34+/CD38- leukemic stem cell-encompassing population was equally sensitive to the combination of PHA-767491 and ABT-737. These results indicate that Bcl-2/Bcl-XL and Mcl-1 act in a redundant fashion as effectors of BMM-mediated AML drug resistance and highlight the potential of Mcl-1-repression to revert BMM-mediated drug resistance in the leukemic stem cell population, thus, prevent disease relapse and ultimately improve patient survival.

Generic substitution of antiretrovirals: patients' and health care providers' opinions.

There is interest in introducing generic antiretroviral drugs (ARVs) into high-income countries in order to maximise efficiency in health care budgets. Studies examining patients' and providers' knowledge and attitudes to generic substitution in HIV are few. This was a cross-sectional, observational study with a convenience sample of adult HIV-infected patients and health care providers (HCPs). Data on demographics, knowledge of generic medicine and facilitators of generic substitution were collected. Descriptive and univariate analysis was performed using SPSS V.23™. Questionnaires were completed by 66 patients. Seventy-one per cent would have no concerns with the introduction of generic ARVs. An increase in frequency of administration (61%) or pill burden (53%) would make patients less likely to accept generic ARVs. There were 30 respondents to the HCP survey. Concerns included the supply chain of generics, loss of fixed dose combinations, adherence and use of older medications. An increase in dosing frequency (76%) or an increase in pill burden (50%) would make HCPs less likely to prescribe a generic ARV. The main perceived advantage was financial. Generic substitution of ARVs would be acceptable to the majority of patients and HCPs. Reinvesting savings back into HIV services would facilitate the success of such a programme.

The Janus Face of Death Receptor Signaling during Tumor Immunoediting.

Cancer immune surveillance is essential for the inhibition of carcinogenesis. Malignantly transformed cells can be recognized by both the innate and adaptive immune systems through different mechanisms. Immune effector cells induce extrinsic cell death in the identified tumor cells by expressing death ligand cytokines of the tumor necrosis factor ligand family. However, some tumor cells can escape immune elimination and progress. Acquisition of resistance to the death ligand-induced apoptotic pathway can be obtained through cleavage of effector cell expressed death ligands into a poorly active form, mutations or silencing of the death receptors, or overexpression of decoy receptors and pro-survival proteins. Although the immune system is highly effective in the elimination of malignantly transformed cells, abnormal/dysfunctional death ligand signaling curbs its cytotoxicity. Moreover, DRs can also transmit pro-survival and pro-migratory signals. Consequently, dysfunctional death receptor-mediated apoptosis/necroptosis signaling does not only give a passive resistance against cell death but actively drives tumor cell motility, invasion, and contributes to consequent metastasis. This dual contribution of the death receptor signaling in both the early, elimination phase, and then in the late, escape phase of the tumor immunoediting process is discussed in this review. Death receptor agonists still hold potential for cancer therapy since they can execute the tumor-eliminating immune effector function even in the absence of activation of the immune system against the tumor. The opportunities and challenges of developing death receptor agonists into effective cancer therapeutics are also discussed.