No abstract

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.

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.

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.

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.

Targeting BCR/ABL with Tyrosine kinase inhibitors (TKIs) is a proven concept for the treatment of Philadelphia chromosome-positive (Ph+) leukemias but the “gatekeeper” mutation T315I confers resistance against all approved TKIs, with the only exception of ponatinib, a multi-targeted kinase inhibitor. Besides resistance to TKIs, T315I also confers additional features to the leukemogenic potential of BCR/ABL, involving endogenous BCR. Therefore we studied the role of BCR on BCR/ABL mutants lacking functional domains indispensable for the oncogenic activity of BCR/ABL. We used the factor independent growth of murine myeloid progenitor 32D cells and the transformation of Rat-1 fibroblasts both mediated by BCR/ABL. Here we report that T315I restores the capacity to mediate factor-independent growth and transformation potential of loss-of-function mutants of BCR/ABL. Targeting endogenous Bcr abrogated the capacity of oligomerization deficient mutant of BCR/ABL-T315I to mediate factor independent growth of 32D cells and strongly reduced their transformation potential in Rat-1 cells, as well as led to the up-regulation of mitogen activated protein kinase (MAPK) pathway.Our data show that the T315I restores the capacity of loss-of-function mutants to transform cells which is dependent on the transphosphorylation of endogenous Bcr, which becomes a putative therapeutic target to overcome resistance by T315I.