Interleukin-7 receptor mutants initiate early T cell precursor leukemia in murine thymocyte progenitors with multipotent potential

Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) exhibits lymphoid, myeloid, and stem cell features and is associated with a poor prognosis. Whole genome sequencing of human ETP-ALL cases has identified recurrent mutations in signaling, histone modification, and hematopoietic development genes but it remains to be determined which of these abnormalities are sufficient to initiate leukemia. We show that activating mutations in the interleukin-7 receptor identified in human pediatric ETP-ALL cases are sufficient to generate ETP-ALL in mice transplanted with primitive transduced thymocytes from p19(Arf-/-) mice. The cellular mechanism by which these mutant receptors induce ETP-ALL is the block of thymocyte differentiation at the double negative 2 stage at which myeloid lineage and T lymphocyte developmental potential coexist. Analyses of samples from pediatric ETP-ALL cases and our murine ETP-ALL model show uniformly high levels of LMO2 expression, very low to undetectable levels of BCL11B expression, and a relative lack of activating NOTCH1 mutations. We report that pharmacological blockade of Jak-Stat signaling with ruxolitinib has significant antileukemic activity in this ETP-ALL model. This new murine model recapitulates several important cellular and molecular features of ETP-ALL and should be useful to further define novel therapeutic approaches for this aggressive leukemia.Louise M. Treanor, Sheng Zhou, Laura Janke, Michelle L. Churchman, Zhijun Ma, Taihe Lu, Shann-Ching Chen, Charles G. Mullighan, Brian P. Sorrentin

Mouse Transplant Models for Evaluating the Oncogenic Risk of a Self-Inactivating XSCID Lentiviral Vector

<div><p></p><p>Hematopoietic stem cell gene therapy requires the use of integrating retroviral vectors in order to stably transmit a therapeutic gene to mature blood cells. Human clinical trials have shown that some vector integration events lead to disrupted regulation of proto-oncogenes resulting in disordered hematopoiesis including T-cell leukemia. Newer vectors have been designed to decrease the incidence of these adverse events but require appropriate pre-clinical assays to demonstrate safety. We have used two distinct mouse serial transplant assays to evaluate the safety of a self-inactivating lentiviral vector intended for use in X-linked severe combined immunodeficiency (XSCID) gene therapy trials. These experiments entailed 28 months of total follow-up and included 386 mice. There were no cases in which the XSCID lentiviral vector clearly caused hematopoietic malignancies, although a single case of B cell malignancy was observed that contained the lentiviral vector as a likely passenger event. In contrast, a SFFV-DsRed γ-retroviral vector resulted in clonal transformation events in multiple secondary recipients. Non-specific pathology not related to vector insertions was noted including T cell leukemias arising from irradiated recipient cells. Overall, this comprehensive study of mouse transplant safety assays demonstrate the relative safety of the XSCID lentiviral vector but also highlight the limitations of these assays.</p></div

Recipient origin of T-cell malignancies arising in the secondary recipients of the EF1a group in experiment 2.

<p>Tumor cells derived from the spleen of transplanted mice were analyzed for CD4 and CD8 expression first (top panels). The abnormal CD4⁺ CD8⁺ leukemic cells were then gated and analyzed for CD45.1 (donor) and CD45.2 (recipient) marker expression. Virtually all CD4⁺CD8⁺ cells exclusively expressed CD45.2 and were therefore derived from the irradiated recipient mice.</p

Malignancy incidence in primary recipient mice in Exp 2 (Donor: CD45.1⁺; Recipient: CD45.2⁺).

<p>Malignancy incidence in primary recipient mice in Exp 2 (Donor: CD45.1⁺; Recipient: CD45.2⁺).</p

Malignancy incidence in secondary recipient mice in Exp. 1 (donor: γ_C^−/−, recipient: γ_C^−/− Rag2^−/− ).

*<p>Other death: Mice died or sacrificed before reaching end points, but not due to hematological malignancies that have transgene insertion (for examples, recipient derived tumor).</p>**<p>All three mice are from the same donor.</p><p>VCN: Vector copy number in peripheral blood mononuclear cells at 30 weeks.</p

Characterization of myeloid malignancies seen in the SFFV-treated secondary recipients.

<p>(A) Hematologic characteristics of myeloid leukemias including peripheral leukocyte counts and spleen weights in individual mice at the time of euthanasia. (B)Flow cytometry analysis of splenic tumor cells from mouse #639. Staining for the Gr1 and Mac1 myeloid markers is shown. Gated normal cells (Gr1⁻ Mac1⁻ ) and tumor cells (Gr1⁺ Mac1⁺) were also analyzed for expression of the DsRed, vector-encoded marker. (C) Peripheral blood smear from a leukemic mouse with abnormal monocytic and granulocytic cells. (D) Southern blot analysis of DNA from bone marrow (B) and spleen (S) of six secondary mice that were derived from two primary recipients is shown. A clonal analysis for vector insertion sites was performed using a single cutter enzyme (BglII) and probed with DsRed cDNA probe. A common clonal pattern was noted from tumors derived from each primary recipient. All mice but #926 had clinical evidence of leukemia, however it was noted that #926 had already converted to a clonal pattern.</p

Skewing of myeloid differentiation in mice transplanted with SFFV-transduced cells.

<p>(A) Percentage of peripheral blood cells in specific hematopoietic lineages at 30 weeks after transplantation. Each dot represents the percent of cells from a given lineage, as determined by flow cytometry, from a single primary transplant recipient from each of the vector groups listed on the X-axis. Statistical comparisons are shown for the indicated groups giving the p value. This analysis demonstrated a significant increase in cells expressing either the Gr1 or the Mac1 marker. (B) Percentage of DsRed+ peripheral blood cells in each lineage in the SFFV group of primary recipients at 30 weeks after transplant. Each line represents the profile from a single mouse and the lineages are indicated on the X-axis.</p