ALBERT

Description: Background: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and is most often of B-cell lineage (Roberts, et al. NEJM 2014). One subtype of B-cell ALL, Philadelphia chromosome-like ALL (Ph-like ALL), is BCR-ABL negative with a gene expression signature similar to that of BCR-ABL positive ALL and is prognostic for poor clinical outcomes (Roberts, et al. NEJM 2014). Ph-like ALL is often associated with rearrangements involving the cytokine receptor-like factor 2 (CRLF2) component of the thymic stromal lymphopoietin receptor (TSLPR) leading to its overexpression (Shochat, et al., JEM 2011). TSLPR is a heterodimer of CRLF2 and IL-7Rα that signals to promote the proliferation and differentiation of B-cell progenitors and acts to promote B-cell transformation in the context of Ph-like ALL (Maude, et al. Blood 2012). Glucocorticoid (GC) therapy plays a central role in the treatment of childhood ALL and resistance to GCs confers a poor prognosis (Piovan, et al. Cancer Cell, 2013). This study examined the role of TSLPR signaling in mediating primary GC resistance and the effects of downstream signal transduction inhibitors to confer GC sensitivity. Methods: Viably cryopreserved splenocytes were obtained from 19 patient-derived xenografts of Ph-like ALL banked in the Children's Oncology Group or Children's Hospital of Philadelphia leukemia biorepositories. Assays were also performed using the Mutz 5 Ph-like ALL cell line. Flow cytometry was used to assess the protein expression of TSLPR and GC receptor (GR). Levels of pERK and pAKT were measured by phosphoflow cytometry at baseline and following TSLP stimulation. Cells were cultured in the presence of 1µM dexamethasone (dex), a GC, with or without 1µM trametinib, a MEK1/2 inhibitor, or 1µM MK2206, a pan-AKT inhibitor, in the presence of TSLP and viability was assessed by Hoechst staining and flow cytometry at 48 hours. Results: Of the 19 Ph-like ALL samples in this study, 11 were CRLF2-rearranged (CRLF2R) and 8 were non-rearranged (CRLF2NR). CRLF2 rearrangements involved P2RY8 or IGH and 9 of 11 samples had concomitant activating mutations in JAK1 or JAK2. CRLF2NRsamples expressed a variety of other translocations involving genes such as JAK2, PDGFR, and ABL1. CRLF2R cells were shown to have significantly greater TSLPR protein expression relative to CRLF2NR cells (p = 0.03). In the presence of TSLP, CRLF2 rearrangement status predicted responsiveness to dex, with CRLF2Rsamples demonstrating significant resistance to dex relative to CRLF2NRsamples (p = 0.004). There was no significant reduction in cell viability following dex treatment in CRLF2R samples (p = 0.5), while dex effectively attenuated cell viability in CRLF2NRsamples (p = 0.008). Importantly, there was no difference in GR expression between these two groups (p = 0.6). CRLF2R samples demonstrated hyperresponsiveness to TSLP stimulation, with a significant induction of pERK and pAKT that exceeded the response of CRLF2NRsamples (p = 0.007 and p = 0.0005, respectively) despite no differences in basal phosphoprotein levels between the two sample groups. Inhibition of MAPK signaling with trametinib or of AKT signaling with MK2206 significantly sensitized CRLF2Rcells to dex in the presence of TSLP when used in combination relative to dex alone (p = 0.0003 and p 〈 0.0001, respectively) and resulted in a significant reduction in cell viability relative to untreated cells (p 〈 0.0001 and p 〈 0.0001, respectively). The Mutz 5 cell line, which expresses both a CRLF2 rearrangement and a JAK2 activating mutation, was used to assess the effect of simultaneous pathway inhibition. Mutz 5 cells were treated with dex alone or dex in combination with trametinib and/or MK2206. While dex alone had no significant effect on cell viability, the addition of trametinib or MK2206 resulted in a 43% and 36% reduction in cell viability, respectively. Furthermore, combined treatment with dex, trametinib, and MK2206 resulted in a 72% reduction in cell viability, demonstrating the efficacy of simultaneous MAPK and PI3K pathway inhibition to confer dex sensitivity. Conclusion: MAPK and PI3K pathway signaling play a role in mediating primary GC resistance in CRLF2R Ph-like ALL, making these pathways potential therapeutic targets for enhancing the efficacy of GC therapy in this patient group. Disclosures Maude: Novartis: Consultancy. Teachey:Novartis: Research Funding.

Description: Glucocorticoids (GCs) are central to the treatment of T-cell acute lymphoblastic leukemia (T-ALL), and upfront resistance to GCs is a poor prognostic factor. We previously demonstrated that over one-third of primary patient T-ALLs are resistant to the GC dexamethasone (DEX) when cultured in the presence of interleukin-7 (IL7), a cytokine that is abundant in the microenvironment of leukemic blasts and that plays a well-established role in leukemogenesis. Mechanistically, we demonstrated that GCs paradoxically induce their own resistance by promoting the upregulation of IL7 receptor (IL7R) expression. In the presence of IL7, this augments signal transduction through the JAK/STAT5 axis, ultimately leading to increased STAT5 transcriptional output. This promotes the upregulation of the pro-survival protein BCL-2, which opposes DEX-induced apoptosis. Given that IL7-induced GC resistance depends on de novo synthesis of IL7R in response to DEX, and that newly synthesized IL7R reaches the cell surface via trafficking through the secretory pathway, we hypothesized that inhibiting the translocation of nascent IL7R peptide into the secretory pathway would effectively overcome IL7-induced DEX resistance. Sec61 is a protein-conducting channel in the membrane of the endoplasmic reticulum (ER) that is required for the cotranslational insertion of nascent polypeptides into the ER upon recognition of the signal sequence on secreted and cell surface proteins. To test the hypothesis that Sec61 inhibition could overcome IL7-induced DEX resistance, we utilized the human T-ALL cell line CCRF-CEM, which recapitulates the resistance phenotype observed in primary patient samples. Using a series of structurally distinct small molecule inhibitors of the Sec61 translocon, we demonstrated that Sec61 inhibition effectively overcomes the increase in cell surface IL7R expression in response to DEX. This occurs despite a persistent elevation in IL7R transcript expression following DEX exposure, confirming that Sec61 inhibitors act post-transcriptionally to attenuate cell surface IL7R expression. To determine whether the sensitivity of IL7R to Sec61 inhibitors is due specifically to the interaction between the IL7R signal sequence and Sec61 inhibitors, we generated IL7R constructs containing hydrophobic amino acid substitutions in the signal sequence, which are predicted to confer resistance to Sec61 inhibitors. Upon transient transfection of these constructs into HEK293T cells, we found that these mutations rendered IL7R resistant to the effects of Sec61 inhibition, confirming that the IL7R signal sequence confers sensitivity to these inhibitors. Using the Bliss independence model of synergy in CCRF-CEM cells, we demonstrated that Sec61 inhibitors potently synergize with DEX to overcome IL7-induced DEX resistance. Importantly, at concentrations at which synergy occurs, Sec61 inhibitors demonstrate no single-agent effect on cell survival, suggesting that these effects are not due to an overall reduction in secretory and membrane protein biogenesis. Furthermore, Sec61 inhibitors failed to sensitize CCRF-CEM cells to other chemotherapies used in T-ALL, none of which demonstrate IL7-induced resistance, thereby suggesting that these effects on DEX sensitivity are due specifically to the reduction in cell surface IL7R. To determine if Sec61 inhibitors prevent the DEX-induced increase in STAT5 transcription, we analyzed BCL-2 expression in cells exposed to DEX and IL7, and found that Sec61 inhibitors attenuate the increase in BCL-2 expression in a dose-dependent manner. We next analyzed a cohort of 34 primary patient T-ALL samples. As in CCRF-CEM cells, we found that specifically in those samples with IL7-induced DEX resistance, Sec61 inhibitors synergized with DEX to induce cell death in the presence of IL7. This effect occurred concomitantly with a reduction in cell surface IL7R expression and BCL-2 expression. Taken together, these data demonstrate the efficacy and feasibility of Sec61 inhibition as a novel and rational therapeutic strategy to overcome the IL7-induced DEX resistance phenotype that affects over one-third of newly diagnosed T-ALL patients. Disclosures Sharp: Kezar Life Sciences: Patents & Royalties. McMinn:Kezar Life Sciences: Employment, Equity Ownership. Kirk:Kezar Life Sciences: Employment, Equity Ownership. Taunton:Global Blood Therapeutics: Equity Ownership; Principia Biopharma: Equity Ownership, Patents & Royalties; Cedilla Therapeutics: Consultancy, Equity Ownership; Pfizer: Research Funding; Kezar Life Sciences: Equity Ownership, Patents & Royalties, Research Funding.

Description: Background: While multi-agent chemotherapy has led to remarkable improvements in survival for children with T-cell acute lymphoblastic leukemia (T-ALL), outcomes remain dismal for the ~ 20% of patients that fail therapy. The goal of this study was to elucidate biological mechanisms contributing to chemoresistance in T-ALL. We hypothesized that chemoresistance owes not only to the aberrant signaling pathways intrinsic to the leukemia cells but also to contributions from an abnormal microenvironment in which they reside. The chemokine receptor ligand pair CXCR4/CXCL12 is important for normal leukocyte trafficking and deregulation is frequently observed in several hematologic malignancies. However, the role this axis plays in T-ALL is largely unknown. Here, we test the hypothesis that CXCR4/CXCL12 creates a sanctuary microenvironment that promotes T-ALL survival and provides protection from chemotherapy. Methods: A panel of T-ALL cell lines and primary patient samples expanded in NOD/SCID/c null (NSG) mice were cultured in the presence or absence of the CXCR4 antagonist, AMD3100, with or without chemotherapy. The human bone marrow stromal cell line, HS27a, which constitutively expresses the CXCR4 ligand CXCL12, was used to recapitulate the tumor microenvironment ex vivo. Transwell migration and modified pseudo-emperipolesis assays were used to examine CXCL12-mediated chemotaxis. Multi-parameter flow cytometry was used to evaluate the impact of activation of the CXCR4/CXCL12 axis on signaling networks, cell survival, and chemotherapy resistance. Results: We found CXCR4 membrane expression on all T-ALL cell lines and xenografts samples tested. Incubation with the CXCR4 ligand CXCL12 resulted in activation of survival signaling cascades (PI3K/AKT and MAPK), an effect blocked with AMD3100. Using a transwell system, we found dose dependent chemotaxis of T-ALL lymphoblasts to CXCL12 that was prevented by AMD3100. T-ALL cells also migrated into HS27a stromal cells in a CXCR4/CXCL12 dependent fashion. In addition, co-culturing T-ALL xenografts cells with HS27a imparted a survival advantage that was promptly eliminated by AMD3100 exposure. To test whether the CXCR4/CXCL12 microenvironment contributed to chemotherapy resistance, T-ALL xenografts cells were co-cultured ex vivo in the presence or absence of HS27a stromal cells and bortezomib or dexamethasone. Stromal cells conferred a marked chemoprotective effect that was specifically blocked by AMD3100. This highlights that stroma-mediated chemoresistance in xenograft samples is in part due to stromal-cell mediated activation of the CXCR4/CXCL12 axis. Conclusions: This study provides evidence for a T-ALL microenvironment that exploits the CXCR4/CXCL12 axis for leukemic cell recruitment, enhanced cell survival, and chemotherapy protection. Our findings also implicate the stroma as a major contributor to chemotherapy resistance in primary expanded patient samples partially due to the activation of the CXCR4/CXCL12 axis. These results further suggest that targeting the stroma through inhibition of this axis may be of therapeutic benefit in patients with chemotherapy resistant T-ALL. Disclosures No relevant conflicts of interest to declare.

Description: Upfront resistance to glucocorticoids (GCs) confers a poor prognosis for children with T-cell acute lymphoblastic leukemia (T-ALL). Using primary diagnostic samples from the Children's Oncology Group trial AALL1231, we previously demonstrated that one-third of patient T-ALL samples are intrinsically resistant to GCs when cultured in the presence of interleukin-7 (IL7), a cytokine that is abundant in the T-ALL microenvironment. Furthermore, we demonstrated that inhibiting JAK/STAT signaling downstream of the IL7 receptor (IL7R) with the JAK1/2 inhibitor ruxolitinib (RUX) overcomes GC resistance in these samples. The objective of the present study was to determine the mechanism of IL7-induced GC resistance in T-ALL and to identify novel therapeutic targets to enhance GC sensitivity. We utilized CCRF-CEM cells, a human T-ALL cell line, as a model system in conjunction with primary patient samples. Exposing CCRF-CEM cells to IL7 induced phosphorylation of STAT5, the predominant downstream effector of IL7R signaling. When cultured in the presence of IL7 and the GC dexamethasone (DEX), CCRF-CEM cells recapitulated the IL7-induced GC resistance phenotype observed in patient samples. This resistance could be overcome with RUX, and Bliss index analysis demonstrated a synergistic relationship between DEX and RUX in the presence of IL7. Furthermore, CRISPR/Cas9 mediated knockout of STAT5 (STAT5 KO) was sufficient to overcome resistance, implicating STAT5 as the critical mediator of IL7-induced GC resistance. DEX exposure potently induced upregulation of IL7R expression in CCRF-CEM cells. Using a luciferase reporter construct containing a series of STAT5 response elements, we demonstrated that in the presence of IL7, DEX-induced upregulation of IL7R expression is associated with increased downstream signal transduction, leading to a significant increase in STAT5 transcriptional output. We then performed RNA-seq to further assess the functional consequences of this enhanced STAT5-mediated transcription. Gene set enrichment analysis (GSEA) revealed that STAT5 target genes were significantly upregulated in cells exposed to DEX and IL7 relative to IL7 alone (normalized enrichment score -2.27; p 〈 0.001; FDR 〈 0.001), suggesting that DEX exposure augments activation of the STAT5 transcriptional program. One critical component of this program that was induced by the combination of DEX and IL7 was the anti-apoptotic family member BCL2, which was not induced by DEX alone. Further analysis of its protein expression in CCRF-CEM cells confirmed this paradoxical upregulation of BCL2 specifically by the combination of DEX and IL7. Furthermore, BCL2 was not upregulated by DEX and IL7 in STAT5 KO cells, consistent with this being a STAT5-mediated effect. IL7-induced GC resistance could be overcome with shRNA-mediated knockdown of BCL2 and with pharmacologic inhibition of BCL2 by venetoclax. Similar to the effect observed with RUX, Bliss index analysis demonstrated synergy between DEX and venetoclax in the presence of IL7. Consistent with our observations in CCRF-CEM cells, an analysis of primary diagnostic T-ALL samples revealed DEX-induced upregulation of IL7R expression in samples with IL7-induced GC resistance, which was associated with increased BCL2 expression in the presence of DEX and IL7. Finally, we performed a similar analysis in healthy murine thymocytes, and found that CD4/CD8 double negative (DN) and CD4 or CD8 single positive (SP) thymocytes, but not double positive (DP) thymocytes, exhibited profound IL7-induced GC resistance that was associated with DEX-induced upregulation of IL7R expression and increased BCL2 expression in the presence of DEX and IL7. These data are consistent with the known role of IL7 specifically at the DN and SP stages of development, and suggests that IL7-induced GC resistance is a physiologic mechanism of GC resistance in normal thymocyte development that is retained during leukemogenesis in a subset of T-ALL samples. Taken together, these data demonstrate that GCs paradoxically induce their own resistance in a subset of T-ALLs and in normal developing T-cells by augmenting a STAT5-mediated pro-survival program that results in upregulation of BCL2. Furthermore, we demonstrate that inhibition of JAK/STAT signaling or of BCL2 may have considerable therapeutic benefit to enhance GC sensitivity in T-ALL patients with IL7-induced GC resistance. Disclosures Teachey: La Roche: Consultancy; Amgen: Consultancy.

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: Although outcomes for patients with T-cell acute lymphoblastic leukemia (T-ALL) have improved dramatically, survival rates for relapsed or refractory T-ALL remain less than 10%. While mechanisms mediating chemotherapy resistance in these patients remain incompletely understood, resistance to glucocorticoids (GC), a central component of therapy, may be particularly important. GC resistance occurs more commonly than resistance to other chemotherapeutic agents in ALL. Additionally, newly diagnosed patients that fail to rapidly clear their peripheral leukemic blasts during an upfront window of prednisone (prednisone poor response) have a poorer outcome, suggesting differences in GC sensitivity may exist at diagnosis. Here, we develop an in vitro assay to model the early prednisone response. Using primary, pre-treatment human T-ALL samples, we demonstrate that individual T-ALLs have distinct intrinsic GC sensitivity thresholds at diagnosis and that this threshold can predict end induction MRD. To interrogate potential mechanisms of GC resistance, we use a panel of patient-derived xenografts (PDX) generated from diagnostic T-ALLs. We find that intrinsic GC resistance is uniformly seen in T-ALLs that arise at the early thymic progenitor (ETP) stage as well as in a subset of non-ETP T-ALLs. Removal of IL7 from the media or inhibition of IL7/JAK/STAT signaling with the JAK1/2 inhibitor ruxolitinib or a novel JAK3 inhibitor, JAK3i, reverses GC resistance in ETP and a subset of the non-ETP T-ALL. This effect is drug specific, since IL7 does not offer protection from death induced by other agents. IL7-dependent GC resistance can be predicted by hyper-responsiveness to IL7 stimulation. Mechanistically, the combination of dexamethasone and ruxolitinib alters the balance between BCL2 and BIM in IL7-dependent, but not IL7-independent, GC resistant T-ALL samples. Together, these data support a model where IL7, a cytokine with leukemogenic properties that is normally present in lymphopoietic niches, contributes to intrinsic GC resistance in a subset of T-ALL samples. This environmentally induced GC resistance may be reversed with IL7/JAK/STAT pathway inhibition. This could result in an augmented leukemotoxic effect of GC treatment, potentially enhancing efficacy of glucocorticoids in a subset of patients and justifying exposure to the toxic side effects of GCs. Disclosures Taunton: Global Blood Therapeutics: Equity Ownership; Kezar Life Sciences: Equity Ownership, Research Funding; Pfizer: Research Funding; Principia Biopharma: Consultancy, Equity Ownership; Circle Pharma: Consultancy, Equity Ownership; Cell Design Labs: Consultancy, Equity Ownership. Wood:Juno: Other: Laboratory Services Agreement; Amgen: Honoraria, Other: Laboratory Services Agreement; Pfizer: Honoraria, Other: Laboratory Services Agreement; Seattle Genetics: Honoraria, Other: Laboratory Services Agreement. Teachey:Novartis: Research Funding.

Description: Background: Survival rates for acute lymphoblastic leukemia (ALL) have risen dramatically but up to 20% of patients relapse and their prognoses are dismal. Resistance to glucocorticoid (GC) agonists is a hallmark of relapsed ALL and a strong predictor of outcome at diagnosis (Dördelmann M et. al, Blood 1999, Schmiegelow K et. al, Leukemia 2001, Tissing WJ et. al, Leukemia 2003). However, the underlying biological pathways that mediate sensitivity to GCs remain to be determined. In this study, we performed a genome-scale shRNA screen to identify mediators of prednisolone sensitivity in ALL cells. Methods: Genome-wide screening was performed using pooled shRNA libraries coupled with next generation sequencing to identify genes that when depleted promote GC sensitivity. Statistical analysis using Bioinformatics for Next Generation Sequencing (BiNGS) and Redundancy and Fold Change (RFC) were employed to identify candidate genes that mediate prednisolone sensitivity (Porter et. al, Leukemia 2012). Validation of hits from the primary screen were performed in Reh and RS4;11 cells. Knockdown of candidate genes MEK2 and MEK4 was determined by western blot. Changes in chemosensitivity upon MEK2 and MEK4 knockdown were determined by Cell Titer-Glo assay (Promega). The levels of apoptotic cells upon chemotherapy treatment in control and knockdown cell lines was determined by Annexin V-PE and 7-Amino-actinomycin D (7AAD) staining (Annexin V-PE Apoptosis Detection Kit, BD Pharmingen, San Diego, CA, USA), followed by flow cytometry using the FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA). The levels of downstream GC target genes including NR3C1, GILZ, and BIM were determined by RT-PCR. The levels of target proteins including GR, pERK, ERK, MEK1, and p53 were determined by western blot. pERK levels in primary matched pairs were determined by multiparameter phosphoflow cytometry. Results: In this study, we performed a genome-scale shRNA screen to identify mediators of prednisolone sensitivity in ALL cell lines. The incorporation of this data with integrated analysis of relapse-specific genetic and epigenetic changes (Hogan et. al, Blood 2012) allowed us to identify the mitogen-activated protein kinase (MAPK) pathway as a mediator of prednisolone resistance in pediatric ALL. Interestingly, depletion of MAPK pathway members, MEK2 and MEK4, increased sensitivity to prednisolone through distinct mechanisms. MEK4 knockdown increased sensitivity specifically to GCs by increasing the mRNA and protein levels of the glucocorticoid receptor (GR). This resulted in greater induction of the GR target genes GILZ and BIM upon prednisolone exposure over time. Importantly, depletion of MEK4 did not affect sensitivity of ALL cells to other chemotherapy agents (doxorubicin, etoposide, and 6-thioguanine). By contrast, MEK2 knockdown increased the sensitivity of cells to each of the chemotherapy agents tested including prednisolone, doxorubicin, etoposide, and 6-thioguanine. Depleting MEK2 decreased activated pERK and increased levels of p53. Over expression of a dominant negative p53 in MEK2 deficient cells reversed sensitivity to doxorubicin and prednisolone, indicating that MEK2 expression mediates chemosenstivity in a p53 dependent manner. Furthermore, inhibition of MEK1/2 pharmacologically with trametinib increased sensitivity of ALL cells to chemotherapy. Trametinib treatment also resulted in increased levels of p53. To determine if activation of the MAPK pathway in patients is associated with recurrent disease we examined seven matched diagnosis and relapse primary samples for MAPK activation as determined by pERK staining, and observed increased pERK levels at relapse in all samples tested. Conclusion: Our data indicate that activation of the MAPK pathway promotes chemoresistance and may drive the development of recurrent disease in pediatric ALL.Asdisrupting MEK2 and MEK4 sensitizes cells to chemotherapy, this makes the MAPK pathway an attractive target for therapeutic intervention in relapsed ALL. Disclosures No relevant conflicts of interest to declare.