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Human CD34 hematopoietic stem and progenitor cells (HSPCs) can reconstitute a human hemato-lymphoid system when transplanted into immunocompromised mice. Although fetal liver-derived and cord blood-derived CD34 cells lead to high engraftment levels, engraftment of mobilized, adult donor-derived CD34 cells has remained poor. We generated so-called MSTRG and MISTRG humanized mice on a background carrying a transgene for human signal regulatory protein α (SIRPα) and human homologs of the cytokine macrophage colony-stimulating factor, thrombopoietin, with or without interleukin-3 and granulocyte-macrophage colony-stimulating factor under murine promoters. Here we transplanted mobilized peripheral blood (PB) CD34 cells in sublethally irradiated newborn and adult recipients. Human hematopoietic engraftment levels were significantly higher in bone marrow (BM), spleen, and PB in newborn transplanted MSTRG/MISTRG as compared with nonobese diabetic/severe combined immunodeficient or human SIRPα-transgenic recipients. Furthermore, newborn transplanted MSTRG/MISTRG mice supported higher engraftment levels of human phenotypically defined HSPCs in BM, T cells in the thymus, and myeloid cells in nonhematopoietic organs such as liver, lung, colon, and skin, approximating the levels in the human system. Similar results were obtained in adult recipient mice. Thus, human cytokine knock-in mice might open new avenues for personalized studies of human pathophysiology of the hematopoietic and immune system in vivo.

Langerhans cell histiocytosis (LCH) is a potentially fatal condition characterized by granulomatous lesions with characteristic clonal mononuclear phagocytes (MNP) harboring activating somatic mutations in MAPK pathway genes, most notably BRAFV600E. We recently discovered that the BRAFV600E mutation can also affect multipotent hematopoietic progenitor cells (HPC) in multisystem LCH disease. How BRAFV600E mutation in HPC leads to LCH is not known. Here we show that enforced expression of the BRAFV600E mutation in early mouse and human multipotent HPC induced a senescence program that led to HPC growth arrest, apoptosis resistance and senescence-associated secretory phenotype (SASP). SASP, in turn, promoted HPC skewing towards the MNP lineage leading to the accumulation of senescent MNP in tissue and the formation of LCH lesions. Accordingly, elimination of senescent cells using INK-ATTAC transgenic mice as well as pharmacologic blockade of SASP improved LCH disease in mice. These results identify senescent cells as a novel target for the treatment of LCH.

Favorable-risk human acute myeloid leukemia (AML) engrafts poorly in currently used immunodeficient mice, possibly because of insufficient environmental support of these leukemic entities. To address this limitation, we here transplanted primary human AML with isolated nucleophosmin (NPM1) mutation and AML with inv(16) in mice in which human versions of genes encoding cytokines important for myelopoiesis (macrophage colony-stimulating factor [M-CSF], interleukin-3, granulocyte-macrophage colony-stimulating factor, and thrombopoietin) were knocked into their respective mouse loci. NPM1 AML engrafted with higher efficacy in cytokine knock-in (KI) mice and showed a trend toward higher bone marrow engraftment levels in comparison with NSG mice. inv(16) AML engrafted with high efficacy and was serially transplantable in cytokine KI mice but, in contrast, exhibited virtually no engraftment in NSG mice. Selected use of cytokine KI mice revealed that human M-CSF was required for inv(16) AML engraftment. Subsequent transcriptome profiling in an independent AML patient study cohort demonstrated high expression of M-CSF receptor and enrichment of M-CSF inducible genes in inv(16) AML cases. This study thus provides a first xenotransplantation mouse model for and informs on the disease biology of inv(16) AML.

Targeting BCR/ABL with tyrosine kinase inhibitors (TKIs) is a proven concept for the treatment of Philadelphia chromosome-positive (Ph+) leukemias. Resistance attributable to either kinase mutations in BCR/ABL or nonmutational mechanisms remains the major clinical challenge. With the exception of ponatinib, all approved TKIs are unable to inhibit the 'gatekeeper' mutation T315I. However, a broad spectrum of kinase inhibition increases the off-target effects of TKIs and may be responsible for cardiovascular issues of ponatinib. Thus, there is a need for more selective options for the treatment of resistant Ph+ leukemias. PF-114 is a novel TKI developed with the specifications of (i) targeting T315I and other resistance mutations in BCR/ABL; (ii) achieving a high selectivity to improve safety; and (iii) overcoming nonmutational resistance in Ph+ leukemias. PF-114 inhibited BCR/ABL and clinically important mutants including T315I at nanomolar concentrations. It suppressed primary Ph+ acute lymphatic leukemia-derived long-term cultures that either displayed nonmutational resistance or harbor the T315I. In BCR/ABL- or BCR/ABL-T315I-driven murine leukemia as well as in xenograft models of primary Ph+ leukemia harboring the T315I, PF-114 significantly prolonged survival to a similar extent as ponatinib. Our work supports clinical evaluation of PF-114 for the treatment of resistant Ph+ leukemia.

The hallmark of Philadelphia chromosome positive (Ph+) leukemia is the BCR/ABL kinase, which is successfully targeted by selective ATP competitors. However, inhibition of BCR/ABL alone is unable to eradicate Ph+ leukemia. The t(9;22) is a reciprocal translocation which encodes not only for the der22 (Philadelphia chromosome) related BCR/ABL, but also for der9 related ABL/BCR fusion proteins, which can be detected in 65% of patients with chronic myeloid leukemia (CML) and 100% of patients with Ph+ acute lymphatic leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96ABL/BCR for the pathogenesis of Ph+ ALL. The co-expression of p96ABL/BCR enhanced the kinase activity and as a consequence, the transformation potential of p185BCR/ABL. Targeting p96ABL/BCR by RNAi inhibited growth of Ph+ ALL cell lines and Ph+ ALL patient-derived long-term cultures (PD-LTCs). Our in vitro and in vivo stem cell studies further revealed a functional hierarchy of p96ABL/BCR and p185BCR/ABL in hematopoietic stem cells. Co-expression of p96ABL/BCR abolished the capacity of p185BCR/ABL to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96ABL/BCR for the pathogenesis of Ph+ ALL.

Key Points BRAFV600E or mutant MAP2K1 expression in human CB CD34+ HSPCs lead to Langerhans cell–like histiocytosis in immune-deficient mice. BRAFV600E-expressing human CB CD34+ HSPCs did not generate hairy cell leukemia in xenograft mouse models.

BackgroundTo develop novel anti-cancer therapeutics we have used a reverse rational approach and searched for human HLA class I molecules known to induce autoimmunity and long-term lasting viral control as a surrogate marker for potential anti-cancer activity. HLA-B*27 or HLA-B*57 are well known genetic factors associated with superior control of viral infections (e.g. HIV and HCV) through processes related to both adaptive and innate immunity. Here we demonstrate that the expression of an optimised HLA-B57-Fc fusion protein (iosH2) exerts anti-tumor efficacy through its multimodal inhibition of LILRB1/2 and KIR3DL1 receptors.Methods iosH2 was produced by stable expression in CHO cells and purified by standard chromatography techniques. Interaction and competition studies were performed using Bio-Layer Interferometry, ELISA, and cell-based assays. Analysis of LILRB1/2 downstream ITIM signaling was assessed using an automated western blot system. Functional cell-based assays including in vitro polarization and phagocytosis of macrophages, T cell and NK cell assays were assessed using live-cell imaging. In vivo efficacy studies were performed using syngeneic and humanized mouse models of cancer.Results iosH2 binds with nanomolar affinity to LILRB1/2 and KIR3DL1, and blocks HLA-G and ANGPTL’s binding to LILRB1/2. iosH2 reduces ITIM downstream signalling including phosphorylation of SHP1/2 and promotes conversion from M2 to M1 macrophage phenotype resulting in enhanced tumor cell phagocytosis in vitro. In addition, iosH2 increases T and NK cell cytotoxicity in co-cultures with cancer cell lines. In vivo efficacy studies demonstrate therapeutic efficacy in syngeneic C38 colon cancer mice and in BRGSF-HIS humanized PDX NSCLC mice in concert with reduction of pro-tumorigenic cytokines.Conclusions iosH2 binds to LILRB1/2 and KIR3DL1, restores immune effector cell function in vitro and demonstrates anti-tumor activity in diverse in vivo mouse models. iosH2 is a first-in-class multi-functional agent that promotes key components of the innate and adaptive immune system leading to profound anti-tumor activity. Clinical development is underway and a phase I trial in preparation.Ethics Approval1. Animal housing and experimental procedures were conducted according to the French and European Regulations and the National Research Council Guide for the Care and Use of Laboratory Animals7–8. The animal facility is authorized by the French authorities (Agreement N° B 21 231 011 EA). All animals procedures (including surgery, anesthesia and euthanasia as applicable) used in the current study (200269/ACT1 C38 SC/Ethical protocol: ONCO 1) were submitted to the Institutional Animal Care and Use Committee of Oncodesign (Oncomet) approved by French authorities (CNREEA agreement N° 91). 2. Animal welfare for this study complies with the UK Animals Scientific Procedures Act 1986 (ASPA) in line with Directive 2010/63/EU of the European Parliament and the Council of 22 September 2010 on the protection of animals used for scientific purposes. All experimental data management and reporting procedures were in strict accordance with applicable Crown Bioscience UK Guidelines and Standard Operating Procedures.