ALBERT

Description: The Philadelphia chromosome (Ph)-like subtype of B-precursor acute lymphoblastic leukemia (ALL) comprises approximately 15% of high-risk ALL, has a kinase-activated gene expression profile similar to that of BCR-ABL1-rearranged ALL, and is associated with a variety of mutations and gene fusions known or predicted to activate oncogenic signal transduction. Children and adults with Ph-like ALL have a very high risk of relapse and poor survival when treated with conventional chemotherapy. Others and we have previously observed constitutive activation of cytokine receptor signaling in Ph-like ALL, particularly of the JAK/STAT and PI3K/Akt/mTOR pathways (Tasian et al., Blood 2012). Preclinical and early clinical studies of JAK inhibition in childhood ALL are in progress. However, the functional role of aberrant PI3K pathway signaling has not been previously investigated in Ph-like ALL. The clinical efficacy of the mTOR inhibitor rapamycin and its analogues has proven suboptimal in various solid and hematologic malignancies, at least in part due to upregulation of Akt signaling, a known sequela of signal transduction inhibitor (STI) monotherapy and a common resistance mechanism. We hypothesized that newer-generation STIs that target multiple PI3K pathway signaling proteins or that selectively inhibit PI3K isoforms may result in superior inhibition of leukemia proliferation and minimize upregulation of alternate signaling pathways. We used patient-derived xenotransplantation models to determine the effects of PI3K pathway STIs upon NOD-SCID-γ-null (NSG) mice well-engrafted with de novo (n = 3) or relapsed (n = 1) childhood Ph-like ALL specimens with JAK2 mutations and/or CRLF2 alterations (Maude et al., Blood 2012). Specifically, we tested the PI3Kα inhibitor BYL719 (30 mg/kg/day), the PI3Kδ inhibitor CAL101 (idelalisib; 30 mg/kg/day), the PI3K/mTOR inhibitor PKI587 (10 mg/kg/day), and the TORC1/TORC2 inhibitor AZD2014 (20 mg/kg/day) to identify the most efficacious PI3K pathway inhibitor(s). Initial pharmacodynamic studies demonstrated that mice treated with each of the four STIs for 72 hours demonstrated potent in vivo inhibition of relevant phosphoproteins in comparison to vehicle-treated mice as measured by phosphoflow cytometric analyses of gated human ALL cells within murine spleens. In particular, both BYL719 and CAL101 treatments resulted in marked inhibition of phosphorylated (p) PI3K, mTOR, S6, and AktS473 via comparison of median fluorescent intensities for STI- vs. vehicle-treated groups with the Mann-Whitney test (p

Description: Key Points 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 patient-derived xenograft models of ETP-ALL.

Description: Introduction: While cure rates for children with acute lymphoblastic leukemia (ALL) are approaching 90% with conventional chemotherapeutic regimens, certain high-risk patient subsets such as early T-cell precursor ALL (ETP-ALL) and Philadelphia Chromosome-like (Ph-like) ALL have an aggressive disease profile and poor prognosis. More recently whole genome and transcriptome sequencing of these high-risk subtypes have revealed several activating gene fusions, alterations and mutations that could result in constitutively activated tyrosine kinases (TKs). Activated TKs are then capable of phosphorylating downstream substrates and impacting several key signaling pathways, resulting in increased cell survival, proliferation and differentiation. Further, the highly heterogeneous nature of these subtypes, along with activating fusions/mutations, makes them refractory to standard chemotherapy. Consequently, there is an urgent need to develop tailored therapeutic strategies for the treatment of these high-risk ALL subtypes. Recent advances in mass-spectrometry and the use of anti-phosphotyrosine antibodies for enrichment of tyrosine phosphorylated peptides have greatly facilitated characterization of the global tyrosine phosphorylation state in cancer cells and identified activated TKs that could be therapeutically targeted. Here we present the first study to quantitatively profile TK activity in xenografted patient biopsies of high-risk pediatric ALL. Methods: In this study, we have established an MS-based phosphotyrosine profiling approach in patient derived xenografts (PDXs) of high-risk pediatric ALL patients and integrated it with a spike-in SILAC quantitative tool to identify and quantify dysregulated TK activity across 16 PDXs. We further extended our study on markedly altered tyrosine phosphorylation in 4 PDXs to assess the therapeutic potential of specific TK inhibitors (TKIs). Immunoblots were performed to validate activated sites and their dephosphorylation upon TKI treatment. RT-PCR and Exome sequencing was carried out to detect novel fusion partners and point mutation sites to validate the activated TK profiles in these PDXs. In vitro cytotoxicity was assessed by mitochondrial metabolic activity assay (Alamar blue) following 48h drug exposures. PDXs were established from ETP-ALL, Ph-like ALL, B-cell precursor (BCP)-ALL, or T-lineage ALL (T-ALL) bone marrow or peripheral blood (PB) biopsies in immune-deficient (NOD/SCID or NSG) mice. Engraftment and in vivo drug responses were assessed by enumeration of the proportion of human versus mouse CD45+cells in the murine PB. Results: Using a quantitative phosphotyrosine profiling method in 16 PDXs, we mapped close to 1900 class I phosphosites with 〉0.75 localization probability and 99% confidence, of which 1394 tyrosine phosphorylated sites had a heavy SILAC partner that allowed quantification. Such profiling could accurately classify the leukemias into either T or B-cell lineages with the high-risk ETP and Ph-like ALL clustering as a distinct group. In particular, PDXs with activated tyrosine phosphorylation profiles of ABL1, FLT3 and JAK were targeted with commercially available TKIs both in vitro and in vivo. Subsequent analysis to investigate the aberrant ABL1 and FLT3 signaling showed a NUP214-ABL1 translocation unique to BCP-ALL in one PDX, and a novel Y572S FLT3 mutation in another. Importantly, using a pre-clinical in vivo xenograft model, the activated JAK-STAT signaling observed in one ETP-ALL PDX was targeted with the JAK1/2 inhibitor, ruxolitinib, leading to a significant decrease in the leukemic blast population in the murine PB. Aberrant ABL1 kinase signaling indicated dasatinib treatment in a Ph+-ALL PDX and a PDX with high phospho-ABL1 (harboring the NUP214-ABL1 translocation), and a complete response and significant progression delay, respectively, were achieved in vivo. Similarly, the uniquely activated FLT3 in one PDX (Y572S mutation) correlated with an in vivoobjective response to the multi-kinase inhibitor sunitinib. Conclusions: This study demonstrates the direct application of an unbiased and quantitative tool to identify aberrant TK signaling in high-risk ALL PDXs and highlights its potential to identify tractable drug targets. This research was supported by NCI NO1CM42216 and by the Australian National Health and Medical Research Council. Disclosures No relevant conflicts of interest to declare.

Description: 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 high-risk 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 〈 .05) in 8 of 8 samples. Enhanced responses to ruxolitinib were observed in samples harboring JAK-activating lesions and higher levels of STAT5 phosphorylation. Rapamycin controlled leukemia burden in all 8 B-ALL samples. Survival analysis of 2 representative B-ALL xenografts demonstrated prolonged survival with rapamycin treatment compared with vehicle (P 〈 .01). These data demonstrate preclinical in vivo efficacy of ruxolitinib and rapamycin in this high-risk B-ALL subtype, for which novel treatments are urgently needed, and highlight the therapeutic potential of targeted kinase inhibition in Ph-like ALL.

Description: Unlike children with single lineage autoimmune cytopenias, children with autoimmune destruction of multiple cell lineages often have chronic therapy-refractory disease. Many patients develop significant morbidity from long-term corticosteroids, and novel therapeutic approaches are needed. We previously demonstrated that treatment with sirolimus (rapamycin) led to complete responses (CRs) in a small cohort of children (5 of 5) with autoimmune lymphoproliferative syndrome (ALPS) in a retrospective case series. We opened a prospective IRB-approved clinical trial treating children with treatment-refractory ALPS with sirolimus. Based on an early efficacy signal, we broadened the inclusion criteria to any children with autoimmune cytopenias who failed or were intolerant to standard immunosuppressive therapy. We have now treated 27 children on the trial. Twelve of the children have ALPS (aged 18 mo. to 18 yrs). Of these, 10 had a durable CR, which was rigorously defined as resolution of autoimmune cytopenias (normal CBC), lymphoproliferation, and other autoimmune manifestations. One child had a near-CR (NCR) with resolution of autoimmunity but mild breakthrough adenopathy with viral illness. One child had a partial response (PR) with improvement in autoimmune disease. This child had only been on therapy for 2 months. Double negative T cells, the biologic hallmark of ALPS, are no longer detectable in 8 of 12 children, suggesting sirolimus targets the pathogenic cell population. In addition to the 12 children on the trial, we have advised clinicians treating an additional 28 children with ALPS at our own institution and at other centers in the USA and from 13 different countries. By report, the majority of these patients (24 of 28) have had complete and durable responses with sirolimus. These additional anecdotal cases support our finding of a 〉80% CR rate on our prospective trial. Many of these children have successfully tolerated sirolimus for over 5 years. Correlative studies in murine models in our laboratory demonstrate mTOR signaling is dysregulated in ALPS, suggesting sirolimus is targeted therapy. Two children with common variable immunodeficiency (CVID) and two with systemic lupus erythematosus (SLE) with refractory multi-lineage autoimmune cytopenias were treated on the trial, and 3 of 4 had a CR. One had NCR with resolution of autoimmune destruction of 2 cell lineages but persistent immune thrombocytopenia (ITP; mean platelet count pre-treatment 50,000/mm3). Six children with Evans syndrome (ES) have been treated on the trial with 3 CR, 1 PR, and 2 non-responders (NRs). We have treated 5 children with refractory single lineage autoimmune cytopenias (4 ITP, 1 autoimmune hemolytic anemia) with 1 CR, 1 PR, and 3 NR. Overall CR rates for the trial are 83% (10 of 12) for ALPS, 60% for non-ALPS multi-lineage autoimmune cytopenias (6 of 10), 20% for single lineage disease (1 of 5), and 63% for the entire cohort (17 of 27). The CR + PR rate for children with multi-lineage autoimmune cytopenias is 91% (20 of 22). Of responding patients, all were steroid-refractory or intolerant and most had failed multiple agents (avg: 3; range 2-6). Side effects were mild and included grade (gr) 1-2 mucositis (10 children; resolved after 1 month in most without therapy modification), gr1 diarrhea (1 child), gr2 acne (2), gr1-2 headache (3), and gr2 hyperlipidemia requiring therapy (2 with fish oil and 1 with statin; all responded). One child had such a rapid reduction in splenomegaly that she developed a splenic infarct potentially attributable to the sirolimus. Studies of immune function were performed on a subset of patients. Remarkably, ALPS patients treated with sirolimus had improvement in immune function and developed normal B and T cell numbers and function. In contrast, patients with non-ALPS immune cytopenias developed mild B and T cell immune suppression, as expected. No child developed any documented serious or opportunistic infection. In summary, sirolimus led to complete and durable responses in a majority of children with multi-lineage autoimmune cytopenias. The responses in ALPS were profound, and these results suggest sirolimus should be considered as first-line for these children based on its efficacy and lack of significant toxicity or immunosuppression. More studies are needed for children with ES, CVID, and SLE; however, initial results of this trial are encouraging. Disclosures: Off Label Use: Sirolimus for autoimmune cytopenias. Smith-Whitley:GlycoMimetics, Inc: Research Funding. Lambert:GSK: Research Funding; Nestle: Consultancy; Amgen: Research Funding.

Description: Key Points Daratumumab is effective against T-ALL in human xenograft models. CD38 is a novel target with broad potential in the treatment of T-ALL.

Description: Key Points Sirolimus monotherapy is a safe and effective steroid-sparing agent, improving autoimmune cytopenias in highly refractory patients. Sirolimus is particularly active in ALPS and should be an early therapy option for patients who require chronic therapy.

Description: Key Points PI3K/mTOR inhibition potently inhibited leukemia proliferation and signal transduction in vivo in human Ph-like ALL xenograft models. Combined PI3K/mTOR and JAK or ABL inhibition was superior to monotherapy in CRLF2/JAK-mutant and ABL/PDGFR-mutant Ph-like ALL models.

Description: Abstract 3251 Recently, an extremely poor risk subset of Philadelphia chromosome (9;22) negative ALL patients was identified that nonetheless have a gene expression profile similar to BCR-ABL positive ALL. The majority of cases in this ALL subset were found to have genetic alterations in IKZF1 (IKAROS). In addition, a significant proportion of these cases had CRLF2 rearrangements, and over half of the CRLF2 rearranged cases also had activating mutations in a JAK kinase. Identifying a biologically-targeted therapy for these patients is important as they are poor responders to conventional chemotherapeutics. Unfortunately, IKZF1 and CRLF2 are not currently targetable, and less than one half of these patients have JAK mutations. mTOR signaling has been shown to be activated in patients with BCR-ABL+ leukemia, in part through an Akt-independent mechanism. Accordingly, mTOR inhibition is effective in BCR-ABL+ ALL, while Akt inhibition is not. As patients with IKZF1 mutations have a gene expression profile similar to BCR-ABL+ ALL, we hypothesized that mTOR may be activated and that targeting mTOR signaling would be effective in this poor risk ALL subtype, whereas targeting Akt would be ineffective. To test this hypothesis, we established xenografts from 21 patients with this poor risk ALL subtype using NOG mice. 17 of 21 samples successfully engrafted with sufficient disease burden to generate second and third generation xenografts. We randomized second-generation xenografts from 4 patient samples to treatment with sirolimus (mTOR inhibitor), perifosine (Akt inhibitor), or vehicle control. Disease was evaluated at weekly intervals by FACS of peripheral blood for human CD19+/CD45+ ALL cells. To assess if there was a differential effect based on JAK mutations and/or CRLF2 rearrangements, we treated mice bearing patient ALL samples from each of the following sub-types: Jak+/CRLF2+, Jak+/CRLF2-, Jak-/CRLF2+, and Jak-/CRLF2-. Mice treated with sirolimus had a profound response with markedly decreased disease in blood and in spleen at sacrifice in all 4 samples (Table 1). Perifosine had no anti-leukemic effect. Both drugs were well tolerated. We confirmed the drugs were hitting target pathways in treated animals by immunoblot for pS6 and pAKT. Sample Avg (range) pb blasts control mice at sacrifice Avg (range) pb blasts sirolimus treated at sacrifice Avg (range) pb blasts perifosine treated sirolimus vs control; perifosine vs control (t-test) Jak-/CRLF2- 5,596 (4,203 – 7,922) 120 (61 – 248) 4,498 (3,522 – 5,474) p = 0.01; p = 0.11 Jak-/CRLF2+ 30,009 (21,173 – 41,166) 12,764 (7,171 – 14,945) 24,587 (12,980 – 44,498) p = 0.01; p = 0.32 Jak+/CRLF2- 50,6730 (24,415 – 90,144) 15,523 (13,814 – 17,915) 31,512 (22,621 – 43,669) p = 0.02; p = 0.10 Jak+/CRLF2+ 6,799 (2,165 – 13,095) 1,676 (398 – 3,431) 5,042 (1,632 – 9,442) p = 0.05; p = 0.31 In conclusion, targeting mTOR with sirolimus maybe an effective treatment for IKZF1 and JAK mutated ALL. Disclosures: No relevant conflicts of interest to declare.

Description: 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.