CRLF2 rearrangements, JAK1/2 point mutations, and JAK2 fusion genes have been identified in Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL), a recently described subtype of pediatric high-risk B-precursor ALL (B-ALL) which exhibits a gene expression profile similar to Ph-positive ALL and has a poor prognosis. Hyperactive JAK/STAT and PI3K/mammalian target of rapamycin (mTOR) signaling is common in this highrisk subset. We, therefore, investigated the efficacy of the JAK inhibitor ruxolitinib and the mTOR inhibitor rapamycin in xenograft models of 8 pediatric B-ALL cases with and without CRLF2 and JAK genomic lesions. Ruxolitinib treatment yielded significantly lower peripheral blast counts compared with vehicle (P < .05) in 6 of 8 human leukemia xenografts and lower splenic blast counts (P < . IntroductionSurvival rates for childhood B-precursor acute lymphoblastic leukemia (B-ALL) approach 90% with current combination chemotherapy regimens. 1 Intensification of chemotherapy regimens has largely been responsible for dramatic improvements in survival; however, recent modifications have yielded diminishing returns, particularly in a subset of leukemias that are relatively resistant to conventional cytotoxic chemotherapy. The identification of underlying genetic alterations in chemotherapy-resistant subtypes, particularly lesions that drive leukemogenesis and can be targeted with novel therapies, remains an urgent need.Genome-wide analyses and next-generation sequencing approaches have advanced our understanding of potential leukemogenic mutations in pediatric ALL. [2][3][4][5][6][7] Recently, these analyses identified a cohort of clinically high-risk pediatric B-precursor ALL with gene expression profiles similar to those of Philadelphia chromosome-positive ALL (Ph ϩ ALL, also termed BCR-ABL1-positive ALL). 2,4,8 This Ph-like cohort suffers high rates of relapse and mortality. The similarity to Ph ϩ ALL suggests that aberrant kinase activity may also drive this subset of ALL. Indeed, several lesions affecting kinase activity and cytokine signaling have recently been identified in Ph-like ALL. 9 Rearrangements in CRLF2 (cytokine receptor-like factor 2), leading to overexpression of this component of the heterodimeric cytokine receptor for thymic stromal lymphopoietin (TSLP), are present in up to 7% of childhood B-precursor ALL overall, 10-12 represent approximately half of Ph-like ALLs, 8 and are highly associated with point mutations in Janus kinase (JAK) family members. 11,13-15 Moreover, CRLF2 overexpression is an independent negative prognostic factor in high-risk pediatric B-ALL. 16 The frequency of genetic alterations in CRLF2 and JAK2 in high-risk B-ALL and Down syndrome-associated ALL 10,17 suggests that these lesions may be key events in leukemogenesis. Consistent with its role in early B-cell development, we have previously demonstrated that TSLP stimulates proliferation of precursor B-ALL cell lines. 18,19 Similarly, JAK signaling has been implicated in BCR-ABL1-mediated transform...

• ETP-ALL, a high-risk subtype of T-ALL, is characterized by aberrant activation of the JAK/STAT signaling pathway.• The JAK1/2 inhibitor ruxolitinib demonstrates robust activity in patientderived xenograft models of ETP-ALL.Early T-cell precursor (ETP) acute lymphoblastic leukemia (ALL) is a recently described subtype of T-ALL characterized by a unique immunophenotype and genomic profile, as well as a high rate of induction failure. Frequent mutations in cytokine receptor and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways led us to hypothesize that ETP-ALL is dependent on JAK/STAT signaling. Here we demonstrate aberrant activation of the JAK/STAT pathway in ETP-ALL blasts relative to non-ETP T-ALL. Moreover, ETP-ALL showed hyperactivation of STAT5 in response to interleukin-7, an effect that was abrogated by the JAK1/2 inhibitor ruxolitinib. In vivo, ruxolitinib displayed activity in 6 of 6 patient-derived murine xenograft models of ETP-ALL, with profound single-agent efficacy in 5 models. Ruxolitinib treatment decreased peripheral blast counts relative to pretreatment levels and compared with control (P < .01) in 5 of 6 ETP-ALL xenografts, with marked reduction in mean splenic blast counts (P < .01) in 6 of 6 samples. Surprisingly, both JAK/STAT pathway activation and ruxolitinib efficacy were independent of the presence of JAK/STAT pathway mutations, raising the possibility that the therapeutic potential of ruxolitinib in ETP-ALL extends beyond those cases with JAK mutations. These findings establish the preclinical in vivo efficacy of ruxolitinib in ETP-ALL, a biologically distinct subtype for which novel therapies are needed. (Blood. 2015;125(11):1759-1767 Introduction Early T-cell precursor (ETP) acute lymphoblastic leukemia (ALL) was first described in 2009 as a subtype of T-ALL with a unique immunophenotype that includes expression of myeloid and early progenitor or stem cell markers in addition to T-cell lineage markers. 1Although overall survival for the majority of T-ALL cases has improved dramatically over the last 50 years, 2 largely due to intensification of chemotherapy regimens, many published studies suggest a large percentage of ETP-ALL cases have dismal outcomes.1,3-7 More recent studies suggest that children with ETP-ALL treated on contemporary protocols that intensify therapy based on minimal residual disease response may not fare as poorly as originally thought. 8,9 ETP-ALL, which represents ;10% to 15% of new T-ALL diagnoses in children 1,4,5 and ;10% to 30% in adults, 3,6 accounts for a disproportionate fraction of T-ALL induction failures (ie, failure to achieve a morphologic remission at the end of the first month of chemotherapy). Novel therapies with alternative mechanistic approaches are urgently needed for chemotherapy-refractory subgroups of ETP-ALL.In addition to an immunophenotype with myeloid/stem cell markers, ETP-ALL cases demonstrate a similar transcriptional and mutational landscape to myeloid leukemias and hematopoietic stem ce...

We have previously demonstrated that mTOR inhibitors (MTIs) are active in preclinical models of acute lymphoblastic leukemia (ALL). MTIs may increase degradation of cyclin D1, a protein involved in dihydrofolate reductase (DHFR) synthesis. Because resistance to methotrexate may correlate with high DHFR expression, we hypothesized MTIs may increase sensitivity of ALL to methotrexate through decreasing DHFR by increasing turn-over of cyclin D1. We tested this hypothesis using multiple ALL cell lines and nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenografted with human ALL. We found MTIs and methotrexate were synergistic in combination in vitro and in vivo. Mice treated with both drugs went into a complete and durable remission whereas single agent treatment caused an initial partial response that ultimately progressed. ALL cells treated with MTIs had markedly decreased expression of DHFR and cyclin D1, providing a novel mechanistic explanation for a combined effect. We found methotrexate and MTIs are an effective and potentially synergistic combination in ALL. (Blood. 2008;112:2020-2023 IntroductionNovel and less toxic treatment strategies are needed for patients with acute lymphoblastic leukemia (ALL). 1 Previously, we have demonstrated that mTOR inhibitors (MTIs), a class of signal transduction inhibitors, are effective as single agents in preclinical models of ALL. 2,3 Combination treatment is the next logical step in the therapeutic use of MTIs. It is important to choose rationallydesigned combinations, building on an understanding of the mechanism of action of MTIs and interactions with other agents.MTIs have been shown to prevent activation and increase degradation of cyclin-dependent kinases, including cyclin D1. 4 Cyclin D1 is involved in Rb phosphorylation and release E2Fs which are involved in dihydrofolate reductase (DHFR) synthesis. [5][6][7] Resistance to methotrexate has been shown in tumors that have high DHFR expression. 8,9 We hypothesized that MTIs may increase the sensitivity of ALL to methotrexate by decreasing cyclin D1, which would in turn would decrease DHFR. 5 We tested this hypothesis using relevant preclinical models. Methods In vitro drug testing using ALL cell linesWe used 9 previously characterized ALL cell lines for these experiments, including 4 murine ALL lines (289, 83, 420, and T309) and 5 human ALL lines (Nalm 6, Nalm16, CEM, Molt-4, and Jurkat; the phenotypes are listed in Table S1, available on the Blood website; see the Supplemental Materials link at the top of the online article). 3 Cell lines were maintained in culture using published techniques. 3 Cells were treated with chemotherapeutic agents, including sirolimus (Wyeth Pharmaceuticals, Philadelphia, PA), temsirolimus (Wyeth), methotrexate (Mayne Pharmaceuticals, Paramus, NJ), L-asparaginase (Merck, Whithouse Station, NJ), doxorubicin (Bedford Labs, Bedford, OH), vincristine (Mayne), dexamethasone (American Regent, Shirley, NY), cytarabine (American Pharmaceutical Partners, Schaumburg, IL), and et...

Patients with autoimmune lymphoproliferative syndrome (ALPS) and systemic lupus erythematosis (SLE) have T-cell dysregulation and produce abnormal, activated T lymphocytes and an atypical peripheral T-cell population, termed double negative T cells (DNTs). T-cell functions, including DNT transition in T-cell development and T-cell activation, are critically dependent on Notch signaling. We hypothesized that inhibiting Notch signaling would be effective in ALPS and SLE by reducing the production of abnormal DNTs and by blocking aberrant T-cell activation. We tested this hypothesis using murine models of ALPS and SLE. Mice were randomized to treatment with the notch pathway inhibitor (gamma-secretase inhibitor), N-S-phenyl-glycine-t-butyl ester (DAPT), or vehicle control. Response to treatment was assessed by measurement of DNTs in blood and lymphoid tissue, by monitoring lymph node and spleen size with ultrasound, by quantifying cytokines by bead-array, by ELISA for total IgG and anti-double-stranded DNA (dsDNA) specific antibodies, and by histopathologic assessment for nephritis. We found a profound and statistically significant decrease in all disease parameters, comparing DAPT-treated mice to controls. Using a novel dosing schema, we avoided the reported toxicities of gamma-secretase inhibitors. Inhibiting the Notch signaling pathway may thus present an effective, novel, and well-tolerated treatment for autoimmune and lymphoproliferative diseases.

Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of abnormal lymphocyte survival caused by defective Fas-mediated apoptosis, leading to lymphadenopathy, hepatosplenomegaly, and an increased number of doublenegative T cells (DNTs). Treatment options for patients with ALPS are limited. Rapamycin has been shown to induce apoptosis in normal and malignant lymphocytes. Since ALPS is caused by defective lymphocyte apoptosis, we hypothesized that rapamycin would be effective in treating ALPS. We tested this hypothesis using rapamycin in murine models of ALPS. We followed treatment response with serial assessment of DNTs by flow cytometry in blood and lymphoid tissue, by serial monitoring of lymph node and spleen size with ultrasonography, and by enzyme-linked immunosorbent assay (ELISA) for anti-double-stranded DNA (dsDNA) antibodies. Three-dimensional ultrasound measurements in the mice correlated to actual tissue measurements at death (r ‫؍‬ .9648). We found a dramatic and statistically significant decrease in DNTs, lymphadenopathy, splenomegaly, and autoantibodies after only 4 weeks when comparing rapamycin-treated mice with controls. Rapamycin induced apoptosis through the intrinsic mitochondrial pathway. We compared rapamycin to mycophenolate mofetil, a second-line agent used to treat ALPS, and found rapamycin's control of lymphoproliferation was superior. We conclude that rapamycin is an effective treatment for murine ALPS and should be explored as treatment for affected humans. (Blood. 2006;108: 1965-1971

ONC201, founding member of the imipridone class of small molecules, is currently being evaluated in advancer cancer clinical trials. We explored single agent and combinatorial efficacy of ONC201 in preclinical models of hematological malignancies. ONC201 demonstrated (GI50 1-8 µM) dose- and time-dependent efficacy in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Burkitt's lymphoma, anaplastic large cell lymphoma (ALCL), cutaneous T-cell lymphoma (CTCL), Hodgkin's lymphoma (nodular sclerosis) and multiple myeloma (MM) cell lines including cells resistant to standard of care (dexamethasone in MM) and primary samples. ONC201 induced caspase-dependent apoptosis that involved activation of the integrated stress response (ATF4/CHOP) pathway, inhibition of Akt phosphorylation, Foxo3a activation, downregulation of cyclin D1, IAP and Bcl-2 family members. ONC201 synergistically reduced cell viability in combination with cytarabine and 5-azacytidine in AML cells. ONC201 combined with cytarabine in a Burkitt's lymphoma xenograft model induced tumor growth inhibition that was superior to either agent alone. ONC201 synergistically combined with bortezomib in MM, MCL and ALCL cells and with ixazomib or dexamethasone in MM cells. ONC201 combined with bortezomib in a Burkitt's lymphoma xenograft model reduced tumor cell density and improved CHOP induction compared to either agent alone. These results serve as a rationale for ONC201 single-agent trials in relapsed/refractory acute leukemia, non-Hodgkin's lymphoma, MM and combination trial with dexamethasone in MM, provide pharmacodynamic biomarkers and identify further synergistic combinatorial regimens that can be explored in the clinic.

No abstract

249 CRLF2 genomic rearrangements that lead to overexpression have been identified in a subset of children with clinically high-risk B-precursor ALL and are highly associated with activating JAK2 mutations (Harvey et al., Blood 2010; Mullighan et al., PNAS 2009 and Nat Genet 2009). These children frequently respond poorly to current intensive cytotoxic chemotherapy regimens and suffer high rates of relapse and mortality. New therapies for these patients are urgently needed. These leukemias exhibit gene expression profiles similar to those of BCR-ABL1 positive ALL, suggesting aberrant kinase activation. We previously demonstrated aberrant JAK/STAT and PI3K/mTOR signaling in CRLF2-overexpressing ALL cell lines and primary human samples in vitro, and thus hypothesize that inhibition of these hyperactive signaling networks has therapeutic relevance. To further characterize this high-risk subset of ALL, we have established multiple xenograft models of CRLF2-rearranged and JAK2-mutated ALL, providing a robust platform for preclinical testing of signal transduction inhibitors. In this model, primary human ALL samples are intravenously injected into NOD/SCID/γc null (NSG) mice, and engraftment is determined by flow cytometry of peripheral blood for human CD19+/CD45+ blasts. Eighteen of 21 primary cryopreserved specimens provided by the Children's Oncology Group engrafted successfully. In order to biochemically characterize the xenografts, we measured phosphorylation of relevant signal transduction proteins by phosphoflow cytometry. Spleens of mice xenografted with CRLF2-rearranged ALL had uniformly increased surface staining of human CRLF2, a component of the heterodimeric receptor complex for the cytokine, thymic stromal lymphopoeitin (TSLP). In vitro stimulation of the CRLF2-overexpressing ALL xenograft spleens with TSLP induced phosphorylation of STAT5, Akt, S6, and 4EBP1, but not of ERK 1/2. In additon, in vitro JAK inhibition with INCB018424 abrogated TSLP-induced JAK/STAT and PI3K/mTOR signaling. The mTOR inhibitor sirolimus, mTORC1/2 inhibitor PP242, and PI3K/mTOR inhibitor PI-103 potently inhibited phosphorylation of S6 and 4EBP1 in these xenograft specimens. These data suggest that the JAK/STAT and PI3K/mTOR pathways may interact in these CRLF2-overexpressing leukemias. These data led us to hypothesize that inhibition of the PI3K/mTOR or JAK/STAT pathways may represent potential therapeutic targets; therefore, we utilized these very high-risk ALL xenograft models to study novel, targeted therapies. Once xenografts had engrafted with sufficient disease burden to detect >5% peripheral CD19+/CD45+ blasts, mice were randomized to receive the mTOR inhibitor sirolimus, the JAK inhibitor INCB018424, or vehicle for three to four weeks. Disease burden was assessed weekly by flow cytometric determination of CD19+/CD45+ blast count in peripheral blood, and at sacrifice, by spleen CD19+/CD45+ blast count. To assess potential differential efficacy based on CRLF2 overexpression (CRLF2+) and/or JAK2 activating mutations (JAK2mut), we treated mice with each subtype of ALL. Sirolimus induced a significant decrease in peripheral blast count in 7 of 7 primary ALL xenografts tested (2 JAK2mut/CRLF2+ samples, 1 JAK2mut/CRLF2- sample, 2 JAK2wt/CRLF2+ samples, and 2 JAK2wt/CRLF2- samples) and a significant decrease in spleen blast count in 6 of 7 samples tested. The most profound reduction of disease burden was seen in the JAK2mut/CRLF2+ leukemias. In addition, the JAK inhibitor INCB018424 decreased peripheral blast count and spleen blast count in a JAK2mut/CRLF2+ xenograft. We next determined if sirolimus conferred a survival advantage in xenografts of 2 ALL specimens, a robust responder and an intermediate responder to sirolimus by blast count. Sirolimus treatment significantly prolonged survival of both xenografts (63 days vs. 23 days, p=0.0015; 91 days vs. 58 days, p=0.0027). Additional human ALL xenograft studies of INCB018424 and other kinase inhibitors are ongoing. The preclinical in vivo efficacy of sirolimus and INCB018424 suggests that novel, targeted therapies have therapeutic potential in CRLF2-overexpressing ALL. Based in part on these data, both INCB018424 and temsirolimus (a parenteral ester of sirolimus) are currently being investigated in multi-center early phase clinical trials for children with relapsed or refractory leukemias. Disclosures: No relevant conflicts of interest to declare.