ALBERT

Description: Children with Down syndrome (DS) are prone to development of high-risk B-cell precursor ALL (DS-ALL), which differs genetically from most sporadic pediatric ALLs. Increased expression of cytokine receptor-like factor 2 (CRLF2), the receptor to thymic stromal lymphopoietin (TSLP), characterizes about half of DS-ALLs and also a subgroup of sporadic “Philadelphia-like” ALLs. To understand the pathogenesis of relapsed DS-ALL, we performed integrative genomic analysis of 25 matched diagnosis-remission and -relapse DS-ALLs. We found that the CRLF2 rearrangements are early events during DS-ALL evolution and generally stable between diagnoses and relapse. Secondary activating signaling events in the JAK-STAT/RAS pathway were ubiquitous but highly redundant between diagnosis and relapse, suggesting that signaling is essential but that no specific mutations are “relapse driving.” We further found that activated JAK2 may be naturally suppressed in 25% of CRLF2pos DS-ALLs by loss-of-function aberrations in USP9X, a deubiquitinase previously shown to stabilize the activated phosphorylated JAK2. Interrogation of large ALL genomic databases extended our findings up to 25% of CRLF2pos, Philadelphia-like ALLs. Pharmacological or genetic inhibition of USP9X, as well as treatment with low-dose ruxolitinib, enhanced the survival of pre-B ALL cells overexpressing mutated JAK2. Thus, somehow counterintuitive, we found that suppression of JAK-STAT “hypersignaling” may be beneficial to leukemic B-cell precursors. This finding and the reduction of JAK mutated clones at relapse suggest that the therapeutic effect of JAK specific inhibitors may be limited. Rather, combined signaling inhibitors or direct targeting of the TSLP receptor may be a useful therapeutic strategy for DS-ALL.

Description: Background: Central nervous system (CNS) involvement by acute lymphoblastic leukemia (ALL) is a major clinical concern. Leukemic cells can be found in the CNS at diagnosis (1-2%) or, more frequently, at relapse (30%). Very little is known about the pathogenesis and therefore there are no targeted therapies. Prophylactic CNS-directed conventional intrathecal chemotherapy or irradiation are required for relapse-free survival. However, they are associated with substantial rates of short and long term toxicity. Therefore, elucidation of molecular mechanisms and pathways mediating leukemia-cell entry and survival in the CNS is needed to develop alternative CNS-directed treatment strategies. Previous studies showed an increased expression of Stearoyl-CoA desaturase (SCD), a key enzyme of the de novo fatty acid synthesis pathway, in B cell precursor (BCP) ALL cells isolated from cerebrospinal fluid (CSF) of patients at the time of CNS relapse. A small SCD positive population was detected in the bone marrow (BM) at leukemia diagnosis in patients who later developed isolated CNS relapse, defining a potential biomarker for CNS relapse. It is unknown, however, if SCD has a functional role in CNS leukemia. Aim: To examine the hypothesis that increased expression of SCD enhances trafficking and survival of human B-ALL cells in the CNS Methods: We analyzed leukemia-cell entry into the CNS using xenografts of human BCP-ALL cell lines. Microarray profile of cells isolated from CNS and BM of transplanted mice was performed. Cell lines were transduced to overexpress human SCD and evaluated in vitro for proliferation kinetics and metabolic SCD activity. In vivo, SCD overexpressing cells were transplanted in NSG mice,sacrificed upon the first symptoms of CNS involvement, e.g. hind limb paralysis. BM, spleen and meninges were collected and analyzed to check human engraftment by FACS. The tumor load was expressed as total amount of leukemic cells in each organ. Competition assays were performed by transplanting SCD overexpressing and WT cells in the same mouse in a 1:1 ratio. Results: BCP-ALL cells transplanted into NSG mice faithfully recapitulated pathological features of meningeal infiltration seen in patients with ALL. Gene expression analysis of cells collected from BM and meninges of leukemic mice revealed up-regulation of the genes belonging to the signaling pathway of sterol regulatory element binding proteins (SREBPs) in ALL cells isolated from the CNS. SCD, whose transcription is controlled by the SREBP family, was significantly upregulated. SCD overexpression did not alter proliferation in vitro. Since SCD introduces a double bond in Stearoyl-CoA, its activity was measured as the ratio of unsaturated/saturated fatty acids in the cells. That ratio was increased in SCD overexpressing cells in vitro, confirming the functionality of the enzyme. In vivo, mice transplanted with SCD overexpressing cells led to a faster onset of CNS disease manifested by a clinical phenotype of earlier hind limb paralysis compared to control and significant increased number of leukemic cells in the CNS (Figure 1A).SCD overexpression also induced CNS engraftment of another B-ALL cell line, REH, which is not naturally prone to invade the central nervous system. Mice transplanted with SCD overexpressing REH cells showed the same phenotype of earlier hind limb paralysis and accumulation of leukemic cells in the CNS as the CNS-prone 018z cells, while WT REH did not show any CNS engraftment but comparable tumor load in BM and spleen (Figure1B). To reproduce the clonal heterogeneity in SCD expression observed previously in patients' BM, we performed a competition assay transplanting SCD overexpressing cells and control cells, expressing different fluorochromes, in the same mouse in a 1:1 ratio. In the CNS, the ratio between SCD overexpressing and WT cells ranged from 2 to 20 fold. This effect was unique to the CNS and not reproducible in the other hematopoietic organs where the 1:1 ratio was maintained (Figure 1C). Moreover, SCD overexpression sensitized leukemic cells to mTOR inhibitors, suggesting a potential therapeutic option Conclusion: SCD has a role in homing and survival of leukemic cells in the CNS and may be used as early predictor of CNS relapse. This study reveals a role for SCD and fatty acid metabolism in the pathogenesis of CNS leukemia suggesting that this pathway maybe targeted for specific therapy of this devastating disease. Figure 1. Figure 1. Disclosures Halsey: Jazz Pharmaceuticals: Honoraria, Other: Support for conference attendance.

Description: Philadelphia-like (Ph-like) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a subgroup of BCP-ALL with an expression pattern similar to BCR-ABL+ BCP-ALL that is associated with poor prognosis. Aberrant expression of CRLF2 in BCP-ALL constitutes the majority of Ph-like BCP-ALL cases. CRLF2 is a receptor subunit that together with the IL7RA subunit comprises the receptor of the proinflammatory cytokine TSLP. Though activation of the IL7R pathway is commonly associated with T-cell malignancies, we previously described IL7RA-activating mutations in BCP-ALL predominantly in the context of CRLF2 aberrant expression (Shochat C. et al. J. Exp. Med. 2011). Here we aimed to test the role of aberrations in CRLF2 and IL7RA in the development of Ph-Like BCP-ALL. Both CRLF2 and TSLP differ extensively between mice and human in amino acid sequence and in lineage expression pattern; loss-of-function germline mutations in IL7RA are associated with lack of B and T cells in mice but with lack of only T cells in humans. Hence, we chose to test the hypothesis that activation of CRLF2/IL7RA contributes to the development of Ph-like BCP-ALL in the context of human lymphopoiesis by using a human xenograft system. To aberrantly activate TSLP/IL7 signaling, we transduced cord-blood (CB) CD34+ hematopoietic progenitors with a set of lentiviral vectors carrying CRLF2 and/or IL7RA [(wild type (IL7RAwt) or IL7R bearing an activating mutation (IL7RAins)] under a B-cell promoter/enhancer (to accentuate B-cell lineage expression). The backbone vector (BB) expressing GFP was used as a control. Transduced CB cells were transplanted into NOD/LtSz-scid IL2Rγnull (NSG) mice and engrafted cells were analyzed 24-30 weeks after transplantation. To test for self-renewal capacity, BM cells from primary engrafted mice were serially transplanted into secondary recipients and the occurrence of human engraftment was tested 24-30 weeks after transplantation. Enforced expression of activated IL7RA with or without CRLF2 led to a significant block in B-cell development at the B-cell progenitor stage (CD19+CD10+sIgM-) in vivo resembling the differentiation stage of leukemic cells (figure Bi). Repertoire sequencing of CD10+CD19+-transduced cells that were sorted from BM of transplanted mice revealed a significantly higher population of DJ-rearranged cells in the CRLF2-IL7RAins-transduced population than in BB-transduced cells (mean ratio of DJ/total rearrangement: BB:0.35+/-0.024, CRLF2-IL7RAins:0.76+/- 0.07, p=0.039, n=3 paired cord blood), in agreement with the early differentiation block phenotype measured by immunophenotyping. These cells furthermore exhibited a Ph-like gene expression pattern when compared to BB-transduced cells in gene set enrichment analysis. Overexpression of IL7RA alone significantly enhanced the early-B fraction (CD19+CD10+CD34+) in the BM of transplanted mice (figure Bii). Additionally, aberrant expression of IL7RA enhanced self-renewal capacity as was evident by an increased ability of the transduced cells to engraft in secondary recipients (number of mice with detectable human engraftment out of secondary transplanted mice: BB:0/6, CRLF2-IL7RAwt:0/3, CRLF2-IL7RAinst:0/3, IL7RAwt-GFP:3/6, IL7RAins-GFP:5/8). Notably, in one case, secondary transplantation of IL7RAins-transduced CB triggered the development of acute BCP-ALL. The leukemic cells (CD19+CD10+CD34+sIgM-) were clonal as validated by V(D)J rearrangement (figure Cii), had the ability to further propagate in serial transplantations and gained secondary Ph-like BCP-ALL-characteristic chromosomal deletions in the short arm of chromosome 9 (in the region including the genes for CDKN2A/B, PAX5 and JAK2) and the short arm of chromosome 7 (the region including IKZF1) (figure Ciii). These results support the hypothesis that aberrant activation of the CRLF2/IL7RA pathway in human B-cell lineage progenitors creates a pre-leukemic state by arresting differentiation of B-cell progenitors, instating Ph-like expression pattern and inducing self-renewal. This is the first model of de novo Ph-like BCP-ALL development from normal human hematopoietic progenitors in vivo. Additionally, we present here a first direct in vivo demonstration of a role for IL7 in human B-cell development. Disclosures No relevant conflicts of interest to declare.

Description: Despite the current risk-based stratification protocol, 15% of pediatric patients with B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) still experience relapse. In the large subset of 'B-other' patients (negative for common fusion transcripts, non-high hyperdiploid and non-Down Syndrome), 'Ph-like' is a high risk subgroup with high incidence of relapses, which represents 30% of B-others or 10-15% of BCP-ALL patients. The PAX5 gene, encoding for a B-cell related transcription factor, is frequently involved in several translocations in Ph-like patients, determining the formation of fusion genes encoding for aberrant proteins. Our previous studies showed that PAX5 fusions sustain survival of leukemic cells by Lymphocyte kinase (LCK) hyperactivation, which can be targeted by the kinase inhibitor Nintedanib/BIBF1120. This study aims (i) to unravel the functional features of PAX5 fusion genes, elucidating the involved signaling pathways; (ii) to develop new pre-clinical strategies to target PAX5 fusion genes, testing the efficacy of the LCK inhibitor BIBF1120. We selected a cohort of 138 B-other cases among 400 childhood BCP-ALL cases enrolled in Italy in AIEOP-BFM ALL2000/R2006 protocols; by gene expression analysis (HG-U133Plus2.0 Affymetrix array), 59/138 presented as Ph-like, and by NGS, a driver fusion gene was identified in 34/59 (58%). Beside known genetic lesions, such as P2RY8/CLRF2 (N=9), EBF1 fusions (N=3), TCF3/HLF (N=1), IKZF1-fusion (N=1) and BCL9/MEF2D (N=1), and single cases with novel fusions, seven cases were carrying a PAX5 fusion gene, representing the most frequent rearrangement. Ex-vivo inhibition with BIBF1120 was setup on primary cells from 5 PAX5-fusion BCP-ALL samples, demonstrating significant efficacy in inducing leukemic cell apoptosis, both as single agent and in combination with standard chemotherapeutic agents (Annexin V viability assay of leukemic cells in co-culture on human bone marrow stroma). Strikingly, in 3/5 cases dexamethasone and BIBF1120 had a synergistic effect and were further tested in in vivo assays. A daily treatment was performed at bulk disease detection (BM aspiration, mean engraftment 20% CD10/CD19+) in patient-derived xenotransplanted NSG mice from 2 different PAX5 fusion cases. At the endpoint (after two weeks), in the PAX5/AUTS2 PDX mice we detected a mild effect in the BM by BIBF1120 alone (disease reduction 24%, p=0.057), further enhanced in combination with dexamethasone (-49%, p=0.005, with a mean engraftment in vehicle mice of 82.6%). In the spleen, the efficacy was highly significant both for BIBF1120 (-52%, p=0.025) and the combination (-91%, p=0.015, mean engraftment vehicle 69.5%). A similar statistical significant effect was observed also in peripheral blood, whilst, BIBF1120 alone showed a specific significant efficacy in CNS meninges. Analogous results have been observed in PDX from the PAX5/DACH2 PDX mice; BM leukemia decreased 47% using BIBF1120 alone (p=0.004), further diminished by the combination (-65%, p=0.0004, with a mean engraftment in vehicle mice of 65%). In the spleen, the efficacy was highly significant both with BIBF1120 (-45.6%, p=0.04) and for the combination (-96.3%, p=0.0008, mean engraftment vehicle 72.4%). Strikingly, BIBF1120 treatment alone showed analogous efficacy in PB and CNS, with leukemia decrease as low as -45% (p=0.04) and -76% (p=0.007), respectively, and the combination nearly achieved remission in PB (-94%, p=0.0001) and it was significant in CNS (-81.2%, p=0.03). Dexamethasone alone was not effective in the BM and spleen, whereas it decreased the leukemia bulk both in PB (-65%, p=0.0004) and CNS (-52.8%, p=0.03). Overall, BIBF1120 treatment was more effective than dexamethasone. Interestingly, phosphoflow analysis showed a marked inhibition by BIBF1120 of pAkt-Thr308 and its downstream effectors, such as pS6 and 4pEBP1, in ex vivo BM and spleen cells. In conclusions, PAX5 fusion genes are highly recurrent among Ph-like patients and can potentially be targeted by Nintendanib/BIBF1120, that showed a significant effect ex vivo and in vivo, even as a single agent. Disclosures No relevant conflicts of interest to declare.

Description: B Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) represents 35% of all cancers in pediatric age group. The cure rate for this disease approaches 90% with current treatment regimens, however only a third of patients with relapse are cured. Therefore, there is an urgent need to focus on subgroups of patients with hallmarks of bad prognosis that could benefit from novel therapeutic approaches. Alterations of Cytokine Receptor-like Factor 2 (CRLF2), a negative prognostic factor in pediatric BCP-ALL, have been identified in up to 10% of patients. However these patients represent half of the high risk Ph-like ALL and of Down Syndrome-associated BCP-ALL. Rearrangements of CRLF2 result in the overexpression of this component of the heterodimeric cytokine receptor for thymic stromal lymphopoietin (TSLP) and is associated with activating mutations of the JAK-STAT pathway. Together these cause hyperactivation of JAK/STAT and PI3K/mTOR signaling. Inhibition of CRLF2/JAK2 signaling has the potential to become a therapeutic targeted intervention for this subgroup of poor prognostic patients. Previous studies have shown that the HDAC inhibitor Givinostat/ITF2357 has potent anti-tumor activity against hematological malignancies, particularly JAK2V617F mutated myeloproliferative neoplasms (MPN) such as polycythemia vera, for which it has already a clinic application and established safety profile. We therefore studied the in vitro and in vivo efficacy of Givinostat in cases with CRLF2 rearrangements. Here we demonstrated that Givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged MHH-CALL4 and MUTZ5 cell lines positive for exon 16 JAK2 mutations. Of note, the observed IC50 values for MHH-CALL4 were lower than those for the SET2 cell line positive control bearing JAK2V617F mutation, both for proliferation (IC50: 0.08±0.05µM vs. 0.14±0.03µM) and apoptosis (IC50: 0.17±0.03µM vs. 0.22±0.04µM). We next investigated the effect of Givinostat on blasts from CRLF2 rearranged BCP-ALL patient samples. For this purpose we developed xenograft models of human CRLF2 rearranged ALL to expand cells from patients and to recapitulate human leukemia in recipient mice. ALL blasts isolated from xenografts were co-cultured on OP9 stroma to perform ex vivo assays. Consistent with our findings in cell lines, Givinostat (0.2µM) reduced the % of live cells (Annexin V/Sytox negative) in all xenografts treated with the drug. In particular, after 72 hours, Givinostat was able to kill up to 〉90% of blast cells in all xenografts in contrast with the vehicle-treated samples which showed 25-60% of blasts still alive after treatment. The induction of cell death in Givinostat treated primografts was confirmed on primary samples from diagnosis using CyTOF which allowed us to observe that CD10+/CRLF2+ blasts were preferentially killed by the drug whereas CD45 high expressing cells (normal residue) remained unaffected by the treatment. Moreover, at low doses (0.2 µM), Givinostat downregulated genes of the JAK/STAT pathway (STAT5A, JAK2, IL7Rα, CRLF2, BCL2L1 and cMYC) and inhibited the basal and ligand induced signaling, reducing the phoshporylation of STAT5 in all tested primografts (mean fold decrease of pSTAT5: 2.4+0.6). Most importantly, to understand if the transcriptional downregulation of CRLF2 resulted in a functional effect, the downmodulation of CRLF2 protein was observed by flow cytometry (mean fold decrease 3.55+1.38). In vivo, Givinostat significantly reduced engraftment of human blasts in xenograft models of CRLF2 positive BCP-ALL (ranging from 1.9 to 34 fold decrease in bone marrow). Furthermore, Givinostat augmented the effect of chemotherapy in inhibiting proliferation and inducing apoptosis in CRLF2 rearranged cell lines and in primografts, in vitro. After 72 hours, the combined treatment reached 4.6-8.8 fold lower % of remaining viable blasts than chemotherapy alone (6.3-35.3% viable cells in chemotherapy-treated samples vs 1.4-4.3% of combination), 2.5-8.5 fold lower than Givinostat alone (4.3-36.4% vs 1.4-4.3%) and 2.4-13 fold lower than Methyl-prednisolone (5.2-39.1 vs 1-16.3%). In conclusion, Givinostat may represent a novel and effective tool, in combination with current chemotherapy, to treat this difficult to handle subset of ALL and these data strongly argue for the translation of Givinostat in combination with conventional therapy into human trials. Disclosures Davis: Fluidigm, Inc: Honoraria. Nolan:Fluidigm, Inc: Equity Ownership.

Description: BACKGROUND: Rearrangements of the CRLF2 gene, present in 7-15% of childhood BCP-ALL, are responsible of the overexpression of Thymic Stromal Lymphopoietin Receptor (TSLPR) and they are correlated with poor prognosis (Chen IM Blood 2012). TSLPR overexpression can be associated with JAK2 mutations, which leads to aberrant activation of JAK/STAT and PI3K/AKT pathways. Although the cross talk of the signaling pathways is still under investigation, there is a rationale for the use of targeted tyrosine kinase inhibitors (TKIs) to treat this subgroup of patients (Maude SL Blood 2012). We focused on the dissection of CRLF2-driven signaling in primary CRLF2 rearranged(r) BCP-ALL samples by using single cell mass cytometry (CyTOF) analysis. We leveraged the high dimensional single cell capability of the CyTOF to understand, with previously unattainable resolution, the activation of these pathways simultaneously in single cells and their response to inhibition with TKIs and anti-TSLPR monoclonal antibodies (mAbs). This revealed heterogeneity in signaling response, identifying subpopulations which differentially activate intracellular signals through TSLPR and differentially respond to ex vivo treatment. METHODS: Twelve BCP-ALL primary samples, 6 CRLF2r and 6 CRLF2 wild type (wt), were investigated and the expression of 24 phenotypic and 15 functional proteins were measured at single cell level using CyTOF as previous described (Bendall SC Science 2011). To assess the response to ex-vivo TSLP stimulation (10ng/mL) and TKIs/mAbs treatment, data were normalized to the basal levels of each phosphoprotein and significance was calculated using student`s t-test. One million cells per condition were treated with different TKIs, Dasatinib, Ruxolitinib and BEZ-235, and two different clones of anti-TSLPR mAbs (130A10 and 130H3) from MRC Technology. RESULTS: As expected, we observed an aberrant TSLP-induced activation of pSTAT5 and prpS6 in CRLF2r patients as compared with CRLF2wt, used as control group (p=0.0055, p= 0.0007). Of note, we also observed a previously not described TSLP-dependent activation of pERK and pCREB (p=0.0313, p=0.0261) suggesting a cross-talk of the TSLPR-driven signaling also with the RAS/MEK pathway. Treatment with TKIs revealed strong inhibitory activity of Dasatinib, which completely inhibited the TSLP-mediated phosphorylation of STAT5, rpS6, CREB and ERK in CRLF2r treated blasts compared to CRLF2r not treated cells (p= 0.0040, 0.0017, 0.0007, 0.0114 respectively). Ruxolitinib, JAK1/2 inhibitor, also reduced rpS6, CREB and ERK phosphorylation (p=0.0025, 0.037, 0.0132). Interestingly one of the two anti-TSLPR tested mAbs (130A10) was also able to significantly inhibit the TSLP-mediated activation of STAT5, rpS6, and ERK (p= 0.0071, 0.0006, 0.0323). Finally, the PI3K/TORk inhibitor, BEZ-235, did not show any statistically significant reductions. Single cell analysis revealed a population of TSLPR overexpressing blasts (range 20-50%) in which the TSLP stimulation resulted in activation of prpS6 but not pSTAT5, present in all the CRLF2r patients. This rpS6 activation could be inhibited by anti-TSLPR mAb, Dasatinib, Ruxolitinib and BEZ-235, except for one patient in which the activation was blunted only by anti-TSLPR mAb and Dasatinib suggesting an activation of prpS6 through a non canonical pathway. This data reveals heterogeneous signaling populations present within this subtype of leukemia driven by TSLPR overexpression. Finally in 3 additional CRLF2r primary samples, we investigated signaling profile of residual blasts (MRD) at Day8 and Day15 post induction initiation. TSLPR expression was consistently maintained in all patients at both time points. Furthermore, residual blasts were still able to respond to TSLP and the induced pSTAT5 could be effectively inhibited by 130A10 anti-TSLPR clone and Ruxolitinib. CONCLUSION: In summary, these data suggest heterogeneity of TSLPR-related signaling with activation of the expected JAK/STAT and PI3K pathways but also RAS/MEK and CREB activation. Further, TSLPR+ blasts exhibit heterogeneous responses to both treatment with TSLP in combination with TKIs or mAb. Finally, the MRD detection by CyTOF allowed the study of the functional activity of the TSLPR positive resistant cells suggesting a role of CRLF2r in the persistence of the leukemic cells and its targeting to treat late and refractory stages of the disease. Disclosures Davis: Fluidigm, Inc: Honoraria. Dyer:Roche Pharmaceuticals: Speakers Bureau; Gilead: Research Funding; ONO Pharmaceuticals: Research Funding. Nolan:Fluidigm, Inc: Equity Ownership.