The Experts below are selected from a list of 1269 Experts worldwide ranked by ideXlab platform
Richard B. Lock - One of the best experts on this subject based on the ideXlab platform.
-
A Novel Engineered Single-Chain Antibody Fragment for Targeting Pediatric Philadelphia Chromosome-like Acute Lymphoblastic Leukemia
Blood, 2020Co-Authors: Sara M.a. Mohamed, Keith C.s. Sia, Richard B. Lock, Karlheinz Friedrich, Andreas Wohlmann, Savvas N. Savvides, Peter R. Schofield, Daniel Christ, Maria Kavallaris, Narges BayatAbstract:Introduction: Philadelphia-like acute lymphoblastic leukaemia (Ph-like ALL) is a high-risk ALL subtype characterized by an inferior survival rate and chemotherapeutic drug resistance (Tasian et al, Blood 130: 2064-2072, 2017). Around 50% of Ph-like ALL cases harbour gene rearrangements leading to the overexpression of cytokine receptor-like factor 2 (CRLF2) (Loh et al, Blood 121: 485-488, 2013). CRLF2 (also known as thymic stromal lymphopoietin receptor, TSLPR) heterodimerizes with the interleukin-7 receptor alpha (IL-7Rα) subunit to form the functional TSLPR. Upon TSLP binding, CRLF2 activates the JAK/STAT signalling pathway, leading to enhanced proliferation and survival of leukemia cells resulting in poor outcomes in patients (Harvey et al, Blood 115: 5312-5321, 2010). The extracellular overexpression of CRLF2 and association with poor prognosis suggest the translational value of designing precision-based therapeutics targeting CRLF2 in Ph-like ALL. Although conventional immunotherapy using full-sized antibodies is a promising treatment strategy that can improve treatment efficiency and minimize off-target toxicity, their clinical translation is challenging due to the high production cost and large size affecting targeting efficiency (Holliger et al, Nat Biotech 23: 1126-1136, 2005). Herein, we validated a novel single-chain variable fragment against CRLF2 (CRLF2-ScFv) for targeting Ph-like ALL cells. Methods: A CRLF2-rearranged Ph-like ALL cell line (MHH-CALL-4) was lentivirally transduced with a CRLF2-targeting shRNA driven by an inducible promoter, enabling the inducible knockdown of CRLF2. CRLF2 expression in MHH-CALL-4 cells after shRNA induction (KD-CALL-4) was evaluated using fluorescence-activated cell sorting (FACS). The cellular association of the CRLF2-ScFv was determined in MHH-CALL-4 and KD-CALL-4 at 4° and 37°C using an indirect immunofluorescence assay. Confocal laser scanning microscopy was used to visualize and compare the cellular association of CRLF2-ScFv in MHH-CALL-4 and KD-CALL-4. The cellular association of CRLF2-ScFv was also investigated ex vivo using a small panel of Ph-like and non-Ph-like ALL patient-derived xenografts (PDXs), representing similar immunophenotype and genetic characteristics to their original disease subtypes (Liem et al, Blood 103: 3905-3914, 2004), and peripheral blood mononuclear cells (PBMCs) to investigate the non-selective association. CRLF2 expression in MHH-CALL-4 and Ph-like ALL PDX cells was quantified using indirect immunofluorescence assay. The downstream impact of CRLF2-ScFv on STAT5 phosphorylation (pSTAT5) was determined by FACS either with or without TSLP stimulation. The statistical significance was tested using Unpaired unequal variances t-test or one-way ANOVA followed by multiple comparisons test. Statistical significance was considered when P ≤ 0.05. All experiments were performed in triplicate. Results: KD-CALL-4 showed a 75% reduction in CRLF2 expression compared with MHH-CALL-4 cells (P = 0.0009). CRLF2-ScFv exhibited a 94% higher association with MHH-CALL-4 compared with KD-CALL-4 cells at 37°C (P = 0.0013). The association of CRLF2-ScFv with MHH-CALL-4 cells was reduced by 75% at the non-proliferating state of cells at 4°C compared to 37°C (P = 0.006). Orthogonally viewed confocal microscopy images showed 82% higher intracellular uptake of CRLF2-ScFv in MHH-CALL-4 compared to KD-CALL-4 cells (P = 0.0003). CRLF2-ScFv showed >80% higher association with a Ph-like PDX sample compared with a control CRLF2low PDX and PBMCs (P < 0.001). Of note, a Ph-like ALL PDX exhibited only one-third of the association with CRLF2-ScFv compared with MHH-CALL-4 cells (P = 0.04), corresponding to the significant difference in CRLF2 surface expression (P = 0.01). CRLF2-ScFv significantly reduced pSTAT5 expression in MHH-CALL-4 cells (P = 0.05) and prevented TSLP-induced STAT5 phosphorylation (P = 0.01), suggesting competition with the TSLP binding site. Conclusion: CRLF2-ScFv is a selective targeting moiety for CRLF2 with a significant internalization potential and receptor antagonistic effect, highlighting the therapeutic implications for targeting Ph-like ALL. Keywords: CRLF2, ScFv, STAT5 phosphorylation, Patient-Derived Xenografts. Disclosures No relevant conflicts of interest to declare.
-
Targeting TSLP-Induced Tyrosine Kinase Signaling Pathways in CRLF2-Rearranged Ph-like ALL.
Molecular cancer research : MCR, 2020Co-Authors: Keith C.s. Sia, Ling Zhong, Chelsea Mayoh, Murray D. Norris, Michelle Haber, Glenn M. Marshall, Mark J. Raftery, Richard B. LockAbstract:Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) is characterized by aberrant activation of signaling pathways and high-risk of relapse. Approximately 50% of Ph-like ALL cases overexpress cytokine receptor-like factor 2 (CRLF2) associated with gene rearrangement. Activated by its ligand thymic stromal lymphopoietin (TSLP), CRLF2 signaling is critical for the development, proliferation and survival of normal lymphocytes. To examine activation of tyrosine kinases regulated by TSLP/CRLF2, phosphotyrosine (P-Tyr) profiling coupled with stable isotope labeling of amino acids in cell culture (SILAC) was conducted using 2 CRLF2-rearranged (CRLF2r) Ph-like ALL cell lines stimulated with TSLP. As a result, increased P-Tyr was detected in previously reported TSLP-activated tyrosine kinases and substrates, including JAK1, JAK2, STAT5, and ERK1/2. Interestingly, TSLP also increased P-Tyr of insulin growth factor 1 receptor (IGF1R) and fibroblast growth factor receptor 1 (FGFR1), both of which can be targeted with small molecule inhibitors. Fixed-ratio combination cytotoxicity assays using the tyrosine kinase inhibitors BMS-754807 and ponatinib that target IGF1R and FGFR1, respectively, revealed strong synergy against both cell line and patient-derived xenograft (PDX) models of CRLF2r Ph-like ALL. Further analyses also indicated off-target effects of ponatinib in the synergy, and novel association of the Ras-associated protein-1 (Rap1) signaling pathway with TSLP signaling in CRLF2r Ph-like ALL. When tested in vivo, the BMS-754807/ponatinib combination exerted minimal efficacy against 2 Ph-like ALL PDXs, associated with low achievable plasma drug concentrations. While this study identified potential new targets in CRLF2r Ph-like ALL, it also highlights that in vivo validation of synergistic drug interactions is essential. Implications: Quantitative phosphotyrosine profiling identified potential therapeutic targets for high-risk CRLF2-rearranged Ph-like ALL
-
targeting tslp induced tyrosine kinase signaling pathways in CRLF2 rearranged ph like all
Molecular Cancer Research, 2020Co-Authors: Keith C.s. Sia, Ling Zhong, Chelsea Mayoh, Murray D. Norris, Michelle Haber, Glenn M. Marshall, Mark J. Raftery, Richard B. LockAbstract:Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) is characterized by aberrant activation of signaling pathways and high risk of relapse. Approximately 50% of Ph-like ALL cases overexpress cytokine receptor-like factor 2 (CRLF2) associated with gene rearrangement. Activated by its ligand thymic stromal lymphopoietin (TSLP), CRLF2 signaling is critical for the development, proliferation, and survival of normal lymphocytes. To examine activation of tyrosine kinases regulated by TSLP/CRLF2, phosphotyrosine (P-Tyr) profiling coupled with stable isotope labeling of amino acids in cell culture (SILAC) was conducted using two CRLF2-rearranged (CRLF2r) Ph-like ALL cell lines stimulated with TSLP. As a result, increased P-Tyr was detected in previously reported TSLP-activated tyrosine kinases and substrates, including JAK1, JAK2, STAT5, and ERK1/2. Interestingly, TSLP also increased P-Tyr of insulin growth factor 1 receptor (IGF1R) and fibroblast growth factor receptor 1 (FGFR1), both of which can be targeted with small-molecule inhibitors. Fixed-ratio combination cytotoxicity assays using the tyrosine kinase inhibitors BMS-754807 and ponatinib that target IGF1R and FGFR1, respectively, revealed strong synergy against both cell line and patient-derived xenograft (PDX) models of CRLF2r Ph-like ALL. Further analyses also indicated off-target effects of ponatinib in the synergy, and novel association of the Ras-associated protein-1 (Rap1) signaling pathway with TSLP signaling in CRLF2r Ph-like ALL. When tested in vivo, the BMS-754807/ponatinib combination exerted minimal efficacy against 2 Ph-like ALL PDXs, associated with low achievable plasma drug concentrations. Although this study identified potential new targets in CRLF2r Ph-like ALL, it also highlights that in vivo validation of synergistic drug interactions is essential. Implication: Quantitative phosphotyrosine profiling identified potential therapeutic targets for high-risk CRLF2-rearranged Ph-like ALL.
Charles G. Mullighan - One of the best experts on this subject based on the ideXlab platform.
-
CRLF2 positive b cell acute lymphoblastic leukemia in adult patients a single institution experience
American Journal of Clinical Pathology, 2017Co-Authors: Sergej Konoplev, Charles G. Mullighan, Nitin Jain, Kathryn G Roberts, Xinyan Lu, Marina Konopleva, Juan Ouyang, Maitrayee Goswami, Marc Valentine, Carlos E BuesoramosAbstract:Objectives Cytokine receptor-like factor 2 ( CRLF2 ) rearrangement is found in approximately 50% of pediatric Ph-like B-cell acute lymphoblastic leukemia (B-ALL), and around 50% of CRLF2 + cases harbor JAK mutations. We analyzed CRLF2 expression and studied its correlation with CRLF2 rearrangement in adult patients with B-ALL. Methods Multiparameter flow cytometry (MFC) was performed consecutively in 126 patients. Results CRLF2 overexpression was detected in 30 (27%) patients, 28 (41%) of 69 patients with B-ALL not otherwise specified, 14 (21%) of 67 untreated patients, and 16 (27%) of 59 patients with relapsed B-ALL, with the highest among Hispanic patients (25/55, 45%). Of CRLF2+ cases, 21 (100%) of 21 cases showed CRLF2 rearrangement by fluorescence in situ hybridization, preferentially involving IGH@CRLF2 (15/15). The entire coding region of JAK2 was sequenced in 14 patients with CRLF2+ B-ALL, and nine (64%) were positive for JAK2 mutations. Conclusions MFC allows a rapid, inexpensive, and reliable detection of B-ALL with CRLF2 rearrangement that would further facilitate testing for JAK2 mutations for targetable therapy.
-
Clinical Implementation of a Testing Algorithm for the Diagnosis of Ph-like B-Cell Acute Lymphoblastic Leukemia
Blood, 2016Co-Authors: Alexandra Reynolds, Charles G. Mullighan, Kathryn G Roberts, Patrick A. Zweidler-mckay, Wang, Sergej Konopleva, Guilin Tang, Zhenya Tang, Jeffrey L. JorgensenAbstract:Introduction: Philadelphia chromosome-like (Ph-like) B acute lymphoblastic leukemia (B-ALL) is characterized by a specific gene expression profile similar to that of Ph positive B-ALL, but lacks the BCR-ABL1 fusion. Ph-like B-ALL is known to frequently carry translocations and/or point mutations in various kinases and/or the cytokine receptor CRLF2 that activate a multi-kinase cascade and is often associated with poorer clinical outcomes. Recent reports have shown that patients with Ph-like B-ALLs respond to ABL1 or JAK2 inhibitors. However, timely diagnosis of Ph-like B-ALL cases remains clinically challenging. Methods: A panel of seven fluorescence in situ hybridization (FISH) probes targeting CRLF2, ABL1, ABL2, JAK2, PDGFRB, CSF1R and EPOR were designed and validated for the detection of rearrangements in Ph-like B-ALL. Of these, FISH for CRLF2was performed on both metaphase and interphase cells and the results were correlated with levels of CRLF2 expression assessed by multiparameter flow cytometry (MFC). Additional comprehensive analysis of fusion transcripts was conducted by PCR based testing. DNA copy number analyses were assessed using SNP microarray and/or MLPA and technologies. Results: For the initial validation, CRLF2 FISH was tested in 10 relapsed and 5 de novo B-ALL cases with CRLF2 overexpression by MFC and 100% concordance was achieved. FISH validation of other probes was successful in detecting known positive rearrangements. Using this panel, we tested a cohort of 57 B-ALL cases with a median age of 31 years old (range 13-81) and unknown Ph-like status, including 14 relapsed and 43 de novo. CRLF2 FISH was positive in all 17 (29.8%) MFC CRLF2 positive cases including 3 co-existing with the BCR-ABL1 fusion, showing 100% concordance. Of the 17 CRLF2 positive cases, five of nine cases tested (55.6%) were positive for JAK2 mutation. IGH-CRLF2 fusion was the most prevalent seen in 10 (58.9%) by metaphase FISH whereas P2RY8-CRLF2 was seen in two (11.8%) cases by metaphase FISH and/or SNP array analysis. One case had a novel fusion PAX5/ZCCHC7-CRLF2 detected by metaphase FISH analysis and the remaining 4 cases showed unknown fusion partners. In 40 (70.2%) CRLF2 negativecases, five (5/57, 8.8%) were positive with our FISH panel including two with CSF1R/PDGFRB, one with JAK2 and one with EPOR rearrangements, while one with NUP214-ABL1fusion was detected by PCR. Conclusions: Using the integrated MFC, FISH and Molecular testing, we demonstrated Ph-like B-ALL in 38.6% (22/57) of unselected B-ALL cases, including 17 CRLF2, 2 CSF1R/PDGFRB, 1 JAK2, 1 EPOR and 1 ABL1 rearrangements. Based on our clinical experience, we further implemented a clinical testing algorithm (Figure 1) for the diagnosis of Ph-like B-ALL. It is noteworthy that BCR-ABL1 fusion and CRLF2 rearrangement are not mutually exclusive and all B-ALL patients should be screened for both rearrangements simultaneously. Disclosures Mullighan:Incyte: Membership on an entity9s Board of Directors or advisory committees; Amgen: Speakers Bureau; Loxo Oncology: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding. Jain:Servier: Consultancy, Honoraria; BMS: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Celgene: Research Funding; Abbvie: Research Funding; Seattle Genetics: Research Funding; Genentech: Research Funding; Infinity: Research Funding; Novartis: Consultancy, Honoraria; Novimmune: Consultancy, Honoraria; ADC Therapeutics: Consultancy, Honoraria, Research Funding; Incyte: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding.
-
aberrant stat5 and pi3k mtor pathway signaling occurs in human CRLF2 rearranged b precursor acute lymphoblastic leukemia
Blood, 2012Co-Authors: Sarah K. Tasian, Cheryl L. Willman, Stephen P. Hunger, Richard C Harvey, Julie M Gastierfoster, Michael J Borowitz, I-ming Chen, Brent L. Wood, Michelle Y. Doral, Charles G. MullighanAbstract:Adults and children with high-risk CRLF2 -rearranged acute lymphoblastic leukemia (ALL) respond poorly to current cytotoxic chemotherapy and suffer unacceptably high rates of relapse, supporting the need to use alternative therapies. CRLF2 encodes the thymic stromal lymphopoietin (TSLP) receptor, which activates cell signaling in normal lymphocytes on binding its ligand, TSLP. We hypothesized that aberrant cell signaling occurs in CRLF2 -rearranged ALL and can be targeted by signal transduction inhibitors of this pathway. In a large number of primary CRLF2 -rearranged ALL samples, we observed increased basal levels of pJAK2, pSTAT5, and pS6. We thus characterized the biochemical sequelae of CRLF2 and JAK alterations in CRLF2 -rearranged ALL primary patient samples via analysis of TSLP-mediated signal transduction. TSLP stimulation of these leukemias further induced robust JAK/STAT and PI3K/mTOR pathway signaling. JAK inhibition abrogated phosphorylation of JAK/STAT and, surprisingly, of PI3K/mTOR pathway members, suggesting an interconnection between these signaling networks and providing a rationale for testing JAK inhibitors in clinical trials. The PI3K/mTOR pathway inhibitors rapamycin, PI103, and PP242 also inhibited activated signal transduction and translational machinery proteins of the PI3K/mTOR pathway, suggesting that signal transduction inhibitors targeting this pathway also may have therapeutic relevance for patients with CRLF2 -rearranged ALL and merit further preclinical testing.
-
Aberrant STAT5 and PI3K/mTOR pathway signaling occurs in human CRLF2 -rearranged B-precursor acute lymphoblastic leukemia
Blood, 2012Co-Authors: Sarah K. Tasian, Cheryl L. Willman, Stephen P. Hunger, Richard C Harvey, Michael J Borowitz, I-ming Chen, Brent L. Wood, Michelle Y. Doral, Julie M. Gastier-foster, Charles G. MullighanAbstract:Adults and children with high-risk CRLF2 -rearranged acute lymphoblastic leukemia (ALL) respond poorly to current cytotoxic chemotherapy and suffer unacceptably high rates of relapse, supporting the need to use alternative therapies. CRLF2 encodes the thymic stromal lymphopoietin (TSLP) receptor, which activates cell signaling in normal lymphocytes on binding its ligand, TSLP. We hypothesized that aberrant cell signaling occurs in CRLF2 -rearranged ALL and can be targeted by signal transduction inhibitors of this pathway. In a large number of primary CRLF2 -rearranged ALL samples, we observed increased basal levels of pJAK2, pSTAT5, and pS6. We thus characterized the biochemical sequelae of CRLF2 and JAK alterations in CRLF2 -rearranged ALL primary patient samples via analysis of TSLP-mediated signal transduction. TSLP stimulation of these leukemias further induced robust JAK/STAT and PI3K/mTOR pathway signaling. JAK inhibition abrogated phosphorylation of JAK/STAT and, surprisingly, of PI3K/mTOR pathway members, suggesting an interconnection between these signaling networks and providing a rationale for testing JAK inhibitors in clinical trials. The PI3K/mTOR pathway inhibitors rapamycin, PI103, and PP242 also inhibited activated signal transduction and translational machinery proteins of the PI3K/mTOR pathway, suggesting that signal transduction inhibitors targeting this pathway also may have therapeutic relevance for patients with CRLF2 -rearranged ALL and merit further preclinical testing.
-
In vivo monitoring of JAK/STAT and PI3K/mTOR signal transduction inhibition in pediatric CRLF2-rearranged acute lymphoblastic leukemia (ALL).
Journal of Clinical Oncology, 2012Co-Authors: Sarah K. Tasian, Shannon L. Maude, Hall, Tiffaney Vincent, Charles G. Mullighan, Cheryl L. Willman, Stephen P. Hunger, Mignon L. Loh, David T. Teachey, Stephan A. GruppAbstract:9506 Background: Therapy intensification for children with B-precursor ALL with high-risk genetic lesions has improved relapse-free survival. CRLF2 rearrangements and JAK2 and IL7RA mutations occur in 10-15% of adult and pediatric ALL patients, most of whom relapse. We and others identified aberrant kinase signatures and perturbed JAK/STAT and PI3K/mTOR signal transduction via in vitro studies of CRLF2-rearranged (CRLF2r) ALLs, suggesting the therapeutic relevance of signal transduction inhibitors (STIs). Our creation of CRLF2r ALL xenograft models has enabled rapid preclinical testing of STIs and measurement of in vivo target inhibition. We hypothesized that inhibition of JAK/STAT and PI3K/mTOR phosphosignaling correlates with therapeutic responses in these models. Methods: NOD/SCID/γc-null (NSG) mice well-engrafted with pediatric ALL samples were treated with the JAK inhibitor ruxolitinib, the mTOR inhibitor sirolimus, or vehicle for 72 hours (for signaling response) or 4 weeks (for therapeutic response...
Glenn M. Marshall - One of the best experts on this subject based on the ideXlab platform.
-
Targeting TSLP-Induced Tyrosine Kinase Signaling Pathways in CRLF2-Rearranged Ph-like ALL.
Molecular cancer research : MCR, 2020Co-Authors: Keith C.s. Sia, Ling Zhong, Chelsea Mayoh, Murray D. Norris, Michelle Haber, Glenn M. Marshall, Mark J. Raftery, Richard B. LockAbstract:Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) is characterized by aberrant activation of signaling pathways and high-risk of relapse. Approximately 50% of Ph-like ALL cases overexpress cytokine receptor-like factor 2 (CRLF2) associated with gene rearrangement. Activated by its ligand thymic stromal lymphopoietin (TSLP), CRLF2 signaling is critical for the development, proliferation and survival of normal lymphocytes. To examine activation of tyrosine kinases regulated by TSLP/CRLF2, phosphotyrosine (P-Tyr) profiling coupled with stable isotope labeling of amino acids in cell culture (SILAC) was conducted using 2 CRLF2-rearranged (CRLF2r) Ph-like ALL cell lines stimulated with TSLP. As a result, increased P-Tyr was detected in previously reported TSLP-activated tyrosine kinases and substrates, including JAK1, JAK2, STAT5, and ERK1/2. Interestingly, TSLP also increased P-Tyr of insulin growth factor 1 receptor (IGF1R) and fibroblast growth factor receptor 1 (FGFR1), both of which can be targeted with small molecule inhibitors. Fixed-ratio combination cytotoxicity assays using the tyrosine kinase inhibitors BMS-754807 and ponatinib that target IGF1R and FGFR1, respectively, revealed strong synergy against both cell line and patient-derived xenograft (PDX) models of CRLF2r Ph-like ALL. Further analyses also indicated off-target effects of ponatinib in the synergy, and novel association of the Ras-associated protein-1 (Rap1) signaling pathway with TSLP signaling in CRLF2r Ph-like ALL. When tested in vivo, the BMS-754807/ponatinib combination exerted minimal efficacy against 2 Ph-like ALL PDXs, associated with low achievable plasma drug concentrations. While this study identified potential new targets in CRLF2r Ph-like ALL, it also highlights that in vivo validation of synergistic drug interactions is essential. Implications: Quantitative phosphotyrosine profiling identified potential therapeutic targets for high-risk CRLF2-rearranged Ph-like ALL
-
targeting tslp induced tyrosine kinase signaling pathways in CRLF2 rearranged ph like all
Molecular Cancer Research, 2020Co-Authors: Keith C.s. Sia, Ling Zhong, Chelsea Mayoh, Murray D. Norris, Michelle Haber, Glenn M. Marshall, Mark J. Raftery, Richard B. LockAbstract:Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) is characterized by aberrant activation of signaling pathways and high risk of relapse. Approximately 50% of Ph-like ALL cases overexpress cytokine receptor-like factor 2 (CRLF2) associated with gene rearrangement. Activated by its ligand thymic stromal lymphopoietin (TSLP), CRLF2 signaling is critical for the development, proliferation, and survival of normal lymphocytes. To examine activation of tyrosine kinases regulated by TSLP/CRLF2, phosphotyrosine (P-Tyr) profiling coupled with stable isotope labeling of amino acids in cell culture (SILAC) was conducted using two CRLF2-rearranged (CRLF2r) Ph-like ALL cell lines stimulated with TSLP. As a result, increased P-Tyr was detected in previously reported TSLP-activated tyrosine kinases and substrates, including JAK1, JAK2, STAT5, and ERK1/2. Interestingly, TSLP also increased P-Tyr of insulin growth factor 1 receptor (IGF1R) and fibroblast growth factor receptor 1 (FGFR1), both of which can be targeted with small-molecule inhibitors. Fixed-ratio combination cytotoxicity assays using the tyrosine kinase inhibitors BMS-754807 and ponatinib that target IGF1R and FGFR1, respectively, revealed strong synergy against both cell line and patient-derived xenograft (PDX) models of CRLF2r Ph-like ALL. Further analyses also indicated off-target effects of ponatinib in the synergy, and novel association of the Ras-associated protein-1 (Rap1) signaling pathway with TSLP signaling in CRLF2r Ph-like ALL. When tested in vivo, the BMS-754807/ponatinib combination exerted minimal efficacy against 2 Ph-like ALL PDXs, associated with low achievable plasma drug concentrations. Although this study identified potential new targets in CRLF2r Ph-like ALL, it also highlights that in vivo validation of synergistic drug interactions is essential. Implication: Quantitative phosphotyrosine profiling identified potential therapeutic targets for high-risk CRLF2-rearranged Ph-like ALL.
Sarah K. Tasian - One of the best experts on this subject based on the ideXlab platform.
-
Outcomes of Patients with Down Syndrome and CRLF2-Overexpressing Acute Lymphoblastic Leukemia (ALL): A Report from the Children's Oncology Group (COG)
Blood, 2020Co-Authors: Karen R. Rabin, Sarah K. Tasian, Kathryn G Roberts, Andrew J Carroll, Meenakshi Devidas, Zhiguo Chen, Yunfeng Dai, Johann K. Hitzler, Nyla A. Heerema, Michael J BorowitzAbstract:Background: Patients with Down syndrome (DS) have an approximately 10-fold increased risk of developing ALL, and the spectrum of genetic alterations differs from that of non-DS ALL. Rearrangement and/or overexpression of cytokine receptor-like factor 2 (CRLF2+) occurs in 50% of DS ALL, compared to only 5-10% CRLF2+ cases in non-DS children and adolescents. JAK2 or JAK1 mutations co-occur in about half of CRLF2+ cases in both DS and non-DS ALL. The prognostic significance of CRLF2+ ALL also appears to differ in the limited data reported to date, with less adverse impact in patients with DS compared to non-DS ALL. Here, we report the clinical characteristics and prognostic significance of B-ALL with CRLF2 overexpression and JAK alterations in children and adolescents/young adults (AYA) with DS who were treated on Children's Oncology Group (COG) clinical trials from 2003-2016. Methods: We analyzed clinical, laboratory, and outcome data for 317 patients with DS B-ALL treated on standard risk (SR) trials AALL0331 and AALL0932 and high risk (HR) trials AALL0232 and AALL1131, for whom CRLF2 status and rearrangement partners (IGH or P2RY8) were ascertained by flow cytometric assessment of surface expression; fluorescence in situ hybridization; and/or polymerase chain reaction (PCR) testing. JAK mutations were ascertained in a subset by PCR and sequencing. Minimal residual disease (MRD) was assessed by flow cytometry at the end of induction (EOI) and at end of consolidation (EOC) for a subset of EOI MRD+ patients. Results: We identified 168/317 (53.0%) CRLF2+ cases, and among those assessed for CRLF2 partner, 17/73 (23.3%) were IGH-CRLF2 and 56/73 (76.7%) were P2RY8-CRLF2. In the subset of 165 cases tested for JAK mutations (85 CRLF2- and 80 CRLF2+), 42/165 (25.4%) had JAK mutations, all of which co-occurred in CRLF2+ cases. CRLF2 positivity was significantly associated with younger age at diagnosis: 140/168 (83.3%) of CRLF2+ cases were under 10 years old, versus 106/149 (71.1%) of CRLF2- cases, p<0.01. P2RY8-CRLF2 cases were significantly more likely than IGH-CRLF2 cases to be associated with age under 10 years at diagnosis (92.9% vs 29.4%, p<0.0001), initial white blood count (WBC) <50x103 (80.4% vs 52.9%, p<0.024), National Cancer Institute (NCI) standard risk status (73.2% vs 23.5%, p<0.0002), and day 29 MRD <0.01% (67.9% vs 37.5%, p<0.028). There were no significant associations between JAK mutation status and any clinical features assessed (sex, initial WBC, central nervous system or testicular involvement, NCI risk group, or EOI/EOC MRD). Among patients with neutral cytogenetics (defined as neither favorable [ETV6-RUNX1, double trisomies of chromosomes 4 and 10] nor unfavorable [BCR-ABL1, KMT2A-rearranged, hypodiploid, iAMP21]), survival did not significantly differ between CRLF2+ and CRLF2- patients (Figure 1A,B), either for 5-year event-free survival (EFS) (79.1 +3.6% vs 77.6 +4.7%, p=0.856) or 5-year overall survival (OS) (89.5 ±2.8% vs 84.9 ±4.1%, p=0.674). There were also no significant differences in EFS or OS based on JAK mutation status or CRLF2 partner, although there were trends toward poorer outcomes in the CRLF2+/JAK+ and IGH-CRLF2 subgroups (Figure 1C). Finally, there was no significant difference in 5-year cumulative incidence of relapse (CIR) according to CRLF2 status (CRLF2+ 16.0 +3.0%, CRLF2- 10.6 +2.6%, p=0.179), although there were non-significant trends toward more relapses in CRLF2+ cases overall and in the NCI HR subgroup analysis. Discussion: Whereas CRLF2 and JAK alterations are associated with higher MRD, poorer survival, and increased CIR in patients with high-risk ALL without DS, these alterations do not demonstrate strong adverse prognostic impact in children and AYAs with DS-ALL treated on recent frontline COG trials, although larger sample sizes are needed to adequately assess for possible poorer prognoses associated with the CRLF2+/JAK+ and IGH-CRLF2 subgroups. Regardless, given the frequency of these targetable lesions and the increased risk of relapse and chemotherapy-associated toxicities in patients with DS-ALL, targeted therapies currently under investigation for these genetic lesions may be beneficial to replace some intensive blocks of therapy in DS-ALL with CRLF2 and/or JAK alterations, both to enhance anti-leukemic efficacy and decrease intensive chemotherapy-associated toxicities. Figure 1 Disclosures Tasian: Gilead Sciences: Research Funding; Incyte Corporation: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Borowitz:Amgen: Honoraria. Mullighan:Illumina: Consultancy, Honoraria, Speakers Bureau; AbbVie, Inc.: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau. Raetz:Celgene: Other: DSMB member; Pfizer: Other: Institutional research funding. Loh:Pfizer: Other: Institutional Research Funding; Medisix Therapeutics: Membership on an entity's Board of Directors or advisory committees. Hunger:Novartis: Consultancy; Amgen: Current equity holder in publicly-traded company, Honoraria.
-
Outcomes of Patients with CRLF2-Overexpressing Acute Lymphoblastic Leukemia without Down Syndrome: A Report from the Children's Oncology Group
Blood, 2020Co-Authors: Sarah K. Tasian, Kathryn G Roberts, Richard C Harvey, Andrew J Carroll, Meenakshi Devidas, Yunfeng Dai, Nyla A. Heerema, I-ming L. Chen, Shalini C. Reshmi, Julie M. Gastier-fosterAbstract:Background: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) occurs in 5-30% of children and adolescents/young adults (AYAs) with B-ALL, is driven by genetic alterations that induce constitutive cytokine receptor and kinase signaling, and is associated with poor clinical outcomes across the older pediatric-to-adult age spectrum (Tasian Blood 2017c, Reshmi Blood 2017, Roberts Blood 2018). Rearrangement and/or overexpression of cytokine receptor-like factor 2 (CRLF2+) occurs in 50% of Ph-like ALL cases with frequently co-occurring JAK2 or JAK1 point mutations or IL7R indel mutations. This study reports the clinical outcomes of children and AYAs with newly-diagnosed National Cancer Institute (NCI) standard-risk (SR) or high-risk (HR) CRLF2+ ALL without Down syndrome treated on four successive Children's Oncology Group (COG) phase 3 clinical trials from 2003 to 2018. Methods: We retrospectively assessed demographic characteristics, laboratory data, and clinical outcomes of 3757 patients with B-ALL treated on COG trials AALL0331 and AALL0932 (SR) and AALL0232 and AALL1131 (HR) whose diagnostic leukemia specimens were analysed by low-density microarray (LDA), fluorescence in situ hybridization, polymerase chain reaction (PCR), and/or anchored multiplex PCR testing (Harvey and Tasian Blood Advances 2020). Minimal residual disease (MRD) was assessed by flow cytometry at the end of induction (EOI) and at the end of consolidation for a subset of EOI MRD+ patients. Results: We identified 77/1541 (5.0%) SR and 244/2216 (11.0%) HR patients with CRLF2+ B-ALL in this cohort. Amongst those with diagnostic leukemia specimens analysed by LDA, 57/72 (79.2%) of SR CRLF2+ and 175/213 (82.2%) of HR CRLF2+ patients were positive for the Ph-like gene expression profile with an 8-gene score ≥0.5. P2RY8-CRLF2 fusions and IGH-CRLF2 translocations were detected in 64/77 (83.1%) and 10/77 (13.0%) of SR CRLF2+ patients and in 98/244 (40.2%) and 103/244 (42.2%) of HR CRLF2+ patients, respectively. CRLF2 rearrangements or F232C mutations were not found in the remaining 3 SR and 43 HR CRLF2+ patients, although other Ph-like alterations were discovered in some (n=3 IGH-EPOR fusions, 1 IL7R indel). Importantly, CRLF2+ vs non-CRLF2-overexpressing (CRLF2-) status was associated with older age (10.8 ±6.5 vs 7.8 ±5.8 years [mean ±SD], p<0.0001), leukocytosis (diagnostic white blood cell count 77.5 ±98.5 vs 49.8 ±119.4 x 10e9/L [mean ±SD], p<0.0001), and higher rates of EOI MRD positivity at a ≥0.01% threshold (47.9% vs 30.1%, p<0.0001), which appeared largely driven by the Ph-like HR cohort as expected (57.9% MRD+ vs 42.1% MRD- in HR CRLF2+ and 44.6% MRD+ vs 55.4% MRD- in SR CRLF2+ patients, p<0.003). Overall, CRLF2+ patients had inferior 5-year event-free survival (EFS; 63.3% ±3.1 vs 82.1% ±0.7, p<0.0001) and overall survival (OS; 79.6% ±2.6 vs 90.5% ±0.6, p<0.0001) compared to CRLF2- patients (Figure 1A-B) and a greater 5-year cumulative incidence of relapse (CIR; 30.4% ±2.7 vs 13.2% ±0.6, p<0.001). While 5-year EFS and OS were particularly poor in Ph-like CRLF2+ HR patients (56.3% ±4.6 and 75.4% ± 3.9, respectively) and non-Ph-like CRLF2+ HR patients (EFS 63.7% ±10.2 and OS 74.4% ±8.9), outcomes for Ph-like CRLF2+ SR (EFS 87.2% ±4.5 and OS 94.5% ±3.1) and non-Ph-like CRLF2+ SR patients (EFS 86.2% ±9.3 and OS 100%) were quite good (p<0.0001 for both EFS and OS; Figure 1C-D). Discussion: Patients with newly-diagnosed CRLF2+ B-ALL treated on frontline COG trials have higher rates of EOI MRD positivity, inferior survival, and increased CIR compared to their CRLF2- counterparts. EFS is especially poor in children and AYAs with NCI HR CRLF2+ ALL, particularly those with the Ph-like expression profile. Conversely, outcomes for children with NCI SR CRLF2+ ALL are reasonably favourable, irrespective of Ph-like status. Development of successful treatment strategies to decrease relapse and to improve survival remains a major therapeutic gap for NCI HR CRLF2+ ALL patients. Current clinical trials are studying the potential efficacy of kinase inhibitor addition to chemotherapy for children, adolescents, and adults with HR Ph-like ALL harboring CRLF2 rearrangements/other JAK pathway alterations or ABL class kinase fusions (NCT0240717, NCT02723994, NCT02883049, NCT03571321). Figure 1 Disclosures Tasian: Gilead Sciences: Research Funding; Incyte Corporation: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Borowitz:Amgen: Honoraria. Mullighan:AbbVie, Inc.: Research Funding; Illumina: Consultancy, Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau. Hunger:Novartis: Consultancy; Amgen: Current equity holder in publicly-traded company, Honoraria. Raetz:Pfizer: Research Funding; Celgene/BMS: Other. Loh:Pfizer: Other: Institutional Research Funding; Medisix Therapeutics: Membership on an entity's Board of Directors or advisory committees.
-
aberrant stat5 and pi3k mtor pathway signaling occurs in human CRLF2 rearranged b precursor acute lymphoblastic leukemia
Blood, 2012Co-Authors: Sarah K. Tasian, Cheryl L. Willman, Stephen P. Hunger, Richard C Harvey, Julie M Gastierfoster, Michael J Borowitz, I-ming Chen, Brent L. Wood, Michelle Y. Doral, Charles G. MullighanAbstract:Adults and children with high-risk CRLF2 -rearranged acute lymphoblastic leukemia (ALL) respond poorly to current cytotoxic chemotherapy and suffer unacceptably high rates of relapse, supporting the need to use alternative therapies. CRLF2 encodes the thymic stromal lymphopoietin (TSLP) receptor, which activates cell signaling in normal lymphocytes on binding its ligand, TSLP. We hypothesized that aberrant cell signaling occurs in CRLF2 -rearranged ALL and can be targeted by signal transduction inhibitors of this pathway. In a large number of primary CRLF2 -rearranged ALL samples, we observed increased basal levels of pJAK2, pSTAT5, and pS6. We thus characterized the biochemical sequelae of CRLF2 and JAK alterations in CRLF2 -rearranged ALL primary patient samples via analysis of TSLP-mediated signal transduction. TSLP stimulation of these leukemias further induced robust JAK/STAT and PI3K/mTOR pathway signaling. JAK inhibition abrogated phosphorylation of JAK/STAT and, surprisingly, of PI3K/mTOR pathway members, suggesting an interconnection between these signaling networks and providing a rationale for testing JAK inhibitors in clinical trials. The PI3K/mTOR pathway inhibitors rapamycin, PI103, and PP242 also inhibited activated signal transduction and translational machinery proteins of the PI3K/mTOR pathway, suggesting that signal transduction inhibitors targeting this pathway also may have therapeutic relevance for patients with CRLF2 -rearranged ALL and merit further preclinical testing.
-
Aberrant STAT5 and PI3K/mTOR pathway signaling occurs in human CRLF2 -rearranged B-precursor acute lymphoblastic leukemia
Blood, 2012Co-Authors: Sarah K. Tasian, Cheryl L. Willman, Stephen P. Hunger, Richard C Harvey, Michael J Borowitz, I-ming Chen, Brent L. Wood, Michelle Y. Doral, Julie M. Gastier-foster, Charles G. MullighanAbstract:Adults and children with high-risk CRLF2 -rearranged acute lymphoblastic leukemia (ALL) respond poorly to current cytotoxic chemotherapy and suffer unacceptably high rates of relapse, supporting the need to use alternative therapies. CRLF2 encodes the thymic stromal lymphopoietin (TSLP) receptor, which activates cell signaling in normal lymphocytes on binding its ligand, TSLP. We hypothesized that aberrant cell signaling occurs in CRLF2 -rearranged ALL and can be targeted by signal transduction inhibitors of this pathway. In a large number of primary CRLF2 -rearranged ALL samples, we observed increased basal levels of pJAK2, pSTAT5, and pS6. We thus characterized the biochemical sequelae of CRLF2 and JAK alterations in CRLF2 -rearranged ALL primary patient samples via analysis of TSLP-mediated signal transduction. TSLP stimulation of these leukemias further induced robust JAK/STAT and PI3K/mTOR pathway signaling. JAK inhibition abrogated phosphorylation of JAK/STAT and, surprisingly, of PI3K/mTOR pathway members, suggesting an interconnection between these signaling networks and providing a rationale for testing JAK inhibitors in clinical trials. The PI3K/mTOR pathway inhibitors rapamycin, PI103, and PP242 also inhibited activated signal transduction and translational machinery proteins of the PI3K/mTOR pathway, suggesting that signal transduction inhibitors targeting this pathway also may have therapeutic relevance for patients with CRLF2 -rearranged ALL and merit further preclinical testing.
-
In vivo monitoring of JAK/STAT and PI3K/mTOR signal transduction inhibition in pediatric CRLF2-rearranged acute lymphoblastic leukemia (ALL).
Journal of Clinical Oncology, 2012Co-Authors: Sarah K. Tasian, Shannon L. Maude, Hall, Tiffaney Vincent, Charles G. Mullighan, Cheryl L. Willman, Stephen P. Hunger, Mignon L. Loh, David T. Teachey, Stephan A. GruppAbstract:9506 Background: Therapy intensification for children with B-precursor ALL with high-risk genetic lesions has improved relapse-free survival. CRLF2 rearrangements and JAK2 and IL7RA mutations occur in 10-15% of adult and pediatric ALL patients, most of whom relapse. We and others identified aberrant kinase signatures and perturbed JAK/STAT and PI3K/mTOR signal transduction via in vitro studies of CRLF2-rearranged (CRLF2r) ALLs, suggesting the therapeutic relevance of signal transduction inhibitors (STIs). Our creation of CRLF2r ALL xenograft models has enabled rapid preclinical testing of STIs and measurement of in vivo target inhibition. We hypothesized that inhibition of JAK/STAT and PI3K/mTOR phosphosignaling correlates with therapeutic responses in these models. Methods: NOD/SCID/γc-null (NSG) mice well-engrafted with pediatric ALL samples were treated with the JAK inhibitor ruxolitinib, the mTOR inhibitor sirolimus, or vehicle for 72 hours (for signaling response) or 4 weeks (for therapeutic response...
Keith C.s. Sia - One of the best experts on this subject based on the ideXlab platform.
-
A Novel Engineered Single-Chain Antibody Fragment for Targeting Pediatric Philadelphia Chromosome-like Acute Lymphoblastic Leukemia
Blood, 2020Co-Authors: Sara M.a. Mohamed, Keith C.s. Sia, Richard B. Lock, Karlheinz Friedrich, Andreas Wohlmann, Savvas N. Savvides, Peter R. Schofield, Daniel Christ, Maria Kavallaris, Narges BayatAbstract:Introduction: Philadelphia-like acute lymphoblastic leukaemia (Ph-like ALL) is a high-risk ALL subtype characterized by an inferior survival rate and chemotherapeutic drug resistance (Tasian et al, Blood 130: 2064-2072, 2017). Around 50% of Ph-like ALL cases harbour gene rearrangements leading to the overexpression of cytokine receptor-like factor 2 (CRLF2) (Loh et al, Blood 121: 485-488, 2013). CRLF2 (also known as thymic stromal lymphopoietin receptor, TSLPR) heterodimerizes with the interleukin-7 receptor alpha (IL-7Rα) subunit to form the functional TSLPR. Upon TSLP binding, CRLF2 activates the JAK/STAT signalling pathway, leading to enhanced proliferation and survival of leukemia cells resulting in poor outcomes in patients (Harvey et al, Blood 115: 5312-5321, 2010). The extracellular overexpression of CRLF2 and association with poor prognosis suggest the translational value of designing precision-based therapeutics targeting CRLF2 in Ph-like ALL. Although conventional immunotherapy using full-sized antibodies is a promising treatment strategy that can improve treatment efficiency and minimize off-target toxicity, their clinical translation is challenging due to the high production cost and large size affecting targeting efficiency (Holliger et al, Nat Biotech 23: 1126-1136, 2005). Herein, we validated a novel single-chain variable fragment against CRLF2 (CRLF2-ScFv) for targeting Ph-like ALL cells. Methods: A CRLF2-rearranged Ph-like ALL cell line (MHH-CALL-4) was lentivirally transduced with a CRLF2-targeting shRNA driven by an inducible promoter, enabling the inducible knockdown of CRLF2. CRLF2 expression in MHH-CALL-4 cells after shRNA induction (KD-CALL-4) was evaluated using fluorescence-activated cell sorting (FACS). The cellular association of the CRLF2-ScFv was determined in MHH-CALL-4 and KD-CALL-4 at 4° and 37°C using an indirect immunofluorescence assay. Confocal laser scanning microscopy was used to visualize and compare the cellular association of CRLF2-ScFv in MHH-CALL-4 and KD-CALL-4. The cellular association of CRLF2-ScFv was also investigated ex vivo using a small panel of Ph-like and non-Ph-like ALL patient-derived xenografts (PDXs), representing similar immunophenotype and genetic characteristics to their original disease subtypes (Liem et al, Blood 103: 3905-3914, 2004), and peripheral blood mononuclear cells (PBMCs) to investigate the non-selective association. CRLF2 expression in MHH-CALL-4 and Ph-like ALL PDX cells was quantified using indirect immunofluorescence assay. The downstream impact of CRLF2-ScFv on STAT5 phosphorylation (pSTAT5) was determined by FACS either with or without TSLP stimulation. The statistical significance was tested using Unpaired unequal variances t-test or one-way ANOVA followed by multiple comparisons test. Statistical significance was considered when P ≤ 0.05. All experiments were performed in triplicate. Results: KD-CALL-4 showed a 75% reduction in CRLF2 expression compared with MHH-CALL-4 cells (P = 0.0009). CRLF2-ScFv exhibited a 94% higher association with MHH-CALL-4 compared with KD-CALL-4 cells at 37°C (P = 0.0013). The association of CRLF2-ScFv with MHH-CALL-4 cells was reduced by 75% at the non-proliferating state of cells at 4°C compared to 37°C (P = 0.006). Orthogonally viewed confocal microscopy images showed 82% higher intracellular uptake of CRLF2-ScFv in MHH-CALL-4 compared to KD-CALL-4 cells (P = 0.0003). CRLF2-ScFv showed >80% higher association with a Ph-like PDX sample compared with a control CRLF2low PDX and PBMCs (P < 0.001). Of note, a Ph-like ALL PDX exhibited only one-third of the association with CRLF2-ScFv compared with MHH-CALL-4 cells (P = 0.04), corresponding to the significant difference in CRLF2 surface expression (P = 0.01). CRLF2-ScFv significantly reduced pSTAT5 expression in MHH-CALL-4 cells (P = 0.05) and prevented TSLP-induced STAT5 phosphorylation (P = 0.01), suggesting competition with the TSLP binding site. Conclusion: CRLF2-ScFv is a selective targeting moiety for CRLF2 with a significant internalization potential and receptor antagonistic effect, highlighting the therapeutic implications for targeting Ph-like ALL. Keywords: CRLF2, ScFv, STAT5 phosphorylation, Patient-Derived Xenografts. Disclosures No relevant conflicts of interest to declare.
-
Targeting TSLP-Induced Tyrosine Kinase Signaling Pathways in CRLF2-Rearranged Ph-like ALL.
Molecular cancer research : MCR, 2020Co-Authors: Keith C.s. Sia, Ling Zhong, Chelsea Mayoh, Murray D. Norris, Michelle Haber, Glenn M. Marshall, Mark J. Raftery, Richard B. LockAbstract:Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) is characterized by aberrant activation of signaling pathways and high-risk of relapse. Approximately 50% of Ph-like ALL cases overexpress cytokine receptor-like factor 2 (CRLF2) associated with gene rearrangement. Activated by its ligand thymic stromal lymphopoietin (TSLP), CRLF2 signaling is critical for the development, proliferation and survival of normal lymphocytes. To examine activation of tyrosine kinases regulated by TSLP/CRLF2, phosphotyrosine (P-Tyr) profiling coupled with stable isotope labeling of amino acids in cell culture (SILAC) was conducted using 2 CRLF2-rearranged (CRLF2r) Ph-like ALL cell lines stimulated with TSLP. As a result, increased P-Tyr was detected in previously reported TSLP-activated tyrosine kinases and substrates, including JAK1, JAK2, STAT5, and ERK1/2. Interestingly, TSLP also increased P-Tyr of insulin growth factor 1 receptor (IGF1R) and fibroblast growth factor receptor 1 (FGFR1), both of which can be targeted with small molecule inhibitors. Fixed-ratio combination cytotoxicity assays using the tyrosine kinase inhibitors BMS-754807 and ponatinib that target IGF1R and FGFR1, respectively, revealed strong synergy against both cell line and patient-derived xenograft (PDX) models of CRLF2r Ph-like ALL. Further analyses also indicated off-target effects of ponatinib in the synergy, and novel association of the Ras-associated protein-1 (Rap1) signaling pathway with TSLP signaling in CRLF2r Ph-like ALL. When tested in vivo, the BMS-754807/ponatinib combination exerted minimal efficacy against 2 Ph-like ALL PDXs, associated with low achievable plasma drug concentrations. While this study identified potential new targets in CRLF2r Ph-like ALL, it also highlights that in vivo validation of synergistic drug interactions is essential. Implications: Quantitative phosphotyrosine profiling identified potential therapeutic targets for high-risk CRLF2-rearranged Ph-like ALL
-
targeting tslp induced tyrosine kinase signaling pathways in CRLF2 rearranged ph like all
Molecular Cancer Research, 2020Co-Authors: Keith C.s. Sia, Ling Zhong, Chelsea Mayoh, Murray D. Norris, Michelle Haber, Glenn M. Marshall, Mark J. Raftery, Richard B. LockAbstract:Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) is characterized by aberrant activation of signaling pathways and high risk of relapse. Approximately 50% of Ph-like ALL cases overexpress cytokine receptor-like factor 2 (CRLF2) associated with gene rearrangement. Activated by its ligand thymic stromal lymphopoietin (TSLP), CRLF2 signaling is critical for the development, proliferation, and survival of normal lymphocytes. To examine activation of tyrosine kinases regulated by TSLP/CRLF2, phosphotyrosine (P-Tyr) profiling coupled with stable isotope labeling of amino acids in cell culture (SILAC) was conducted using two CRLF2-rearranged (CRLF2r) Ph-like ALL cell lines stimulated with TSLP. As a result, increased P-Tyr was detected in previously reported TSLP-activated tyrosine kinases and substrates, including JAK1, JAK2, STAT5, and ERK1/2. Interestingly, TSLP also increased P-Tyr of insulin growth factor 1 receptor (IGF1R) and fibroblast growth factor receptor 1 (FGFR1), both of which can be targeted with small-molecule inhibitors. Fixed-ratio combination cytotoxicity assays using the tyrosine kinase inhibitors BMS-754807 and ponatinib that target IGF1R and FGFR1, respectively, revealed strong synergy against both cell line and patient-derived xenograft (PDX) models of CRLF2r Ph-like ALL. Further analyses also indicated off-target effects of ponatinib in the synergy, and novel association of the Ras-associated protein-1 (Rap1) signaling pathway with TSLP signaling in CRLF2r Ph-like ALL. When tested in vivo, the BMS-754807/ponatinib combination exerted minimal efficacy against 2 Ph-like ALL PDXs, associated with low achievable plasma drug concentrations. Although this study identified potential new targets in CRLF2r Ph-like ALL, it also highlights that in vivo validation of synergistic drug interactions is essential. Implication: Quantitative phosphotyrosine profiling identified potential therapeutic targets for high-risk CRLF2-rearranged Ph-like ALL.