3D Modeling of Novel Transforming JAK Mutations in T-Cell Acute Lymphoblastic Leukemia Reveals Altered Pseudokinase-Kinase Domain Interactions That Result in Constitutive JAK Kinase Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 869-869
Author(s):  
Kirsten Canté-Barrett ◽  
Joost CM Uitdehaag ◽  
Jessica GCAM Buijs-Gladdines ◽  
Wilco K Smits ◽  
Rogier C Buijsman ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
3D Model ◽  
Kinase Activity ◽  
Lymphoblastic Leukemia ◽  
Kinase Domain ◽  
Salt Bridges ◽  
Wild Type ◽  
Jak Kinase ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Equity Ownership

Abstract Background: Many pediatric T-cell acute lymphoblastic leukemia patients harbor mutations in IL7Ra or downstream molecules encoded by JAK1, JAK3, N-RAS, K-RAS, NF1, AKT, and PTEN. These mutated signaling molecules can contribute to leukemia by disturbing a multitude of cellular processes such as the cell cycle, epigenetics, apoptosis, or affecting other important signal transduction pathways. Aims: We aimed to determine the overall incidence of JAK family mutations in a large cohort of T-ALL patients. We also aimed to generate a 3D JAK1 model including known and newly identified JAK mutations in order to better understand how these contribute to JAK kinase activity and the transformation of cells. Methods: We screened 146 pediatric T-ALL patient samples for mutations in the FERM, pseudokinase and kinase domains of the Janus kinase gene family (JAK1-3, TYK2). To establish a 3D JAK1 model, we superimposed individual pseudokinase and kinase crystallographic structures on the homologous TYK2 pseudokinase-kinase structure. We visualized JAK mutations and their effects on the 3D structure. We modified the IL3-dependent Ba/F3 cell line to express JAK mutant or wild type genes upon induction by doxycycline. We tested these Ba/F3 derivative lines for transforming ability, signaling, and resistance to various inhibitors in the absence of IL3. Results: JAK1 or JAK3 mutations were found in 10 patients; no mutations were found in JAK2 or TYK2. We found JAK1 and JAK3 mutations as previously reported, but also identified amino acid substitutions as a result of novel JAK1 mutations including V427M, L624YPILKV, E668Q, P815S, and T901G. Our novel 3D model of JAK1 places most mutations in one of two crucial pseudokinase-kinase interaction sites, which can weaken the interaction and facilitate constitutive kinase activity. One interaction is between the hinge region of the pseudokinase domain and the loop in the kinase domain, which is supported by four salt bridges. Mutations in T-ALL disrupting these salt bridges include E668Q, R724H and its JAK3 equivalent R657Q, and T901G. The second interaction with the kinase domain is formed by a helical domain in the pseudokinase domain, located just upstream of the conserved F575-F636-V658 triad. This F-F-V triad is predicted to act as a structural switch that controls the catalytic activity of JAK kinases. Various mutations occur in the direct vicinity and can affect the function of this switch. V658F in T-ALL and its JAK2 equivalent V617F in polycythemia vera patients are mutations in this triad. The frequent JAK3 mutation M511I in T-ALL flanks the F513 residue (equivalent of JAK1 F575) and also affects the F-F-V triad. The L624YPILKV insertion mutation is located in a loop near the helical domain, which may also subtly compromise the F-F-V triad structural switch leading to derepression of the kinase domain. Expression of mutant JAK genes-in contrast to the wild type genes-transforms Ba/F3 cells by supporting IL3-independent growth, and by activating downstream RAS-MEK-ERK and PI3K-AKT pathways. This pathway activation as a result of ligand-independent mutant JAK kinase activity was confirmed by measuring phospho-proteins including p-MEK, p-ERK, p-AKT, p-mTOR, and p-p70S6K, and can be blocked by JAK inhibitors. Notably, JAK3 mutants signal significantly weaker than JAK1 mutants, possibly due to different dependence on (endogenous) receptors that normally mediate wild type JAK signaling. Summary/Conclusion: In a 3D model, we show that JAK mutations are located in critical interface regions between the pseudokinase and kinase domains, maintaining the kinase in an open, active confirmation. The inducible Ba/F3 model system confirms the transforming capacity of JAK mutations, reveals constitutive active downstream signaling, and is also suitable to test the effect of various inhibitors. The visualization of various JAK mutations in a 3D model and how these contribute to kinase activity provides insight in how mutant JAK could be inhibited, helping guide the development of new small molecule inhibitors of mutant JAKs. Disclosures Buijsman: Netherlands Translational Research Center B.V.: Equity Ownership, Other: founder and shareholder. Zaman:Netherlands Translational Research Center B.V.: Equity Ownership, Other: founder and shareholder.

T-Cell Acute Lymphoblastic Leukemia Patients with Mutations in IL7Ra or Downstream RAS-MEK or PI3K-AKT Can be Collectively Targeted By Combination of RAS and AKT Inhibitors

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 445-445
Author(s):  
Kirsten Canté-Barrett ◽  
Jill AP Spijkers-Hagelstein ◽  
Jessica GCAM Buijs-Gladdines ◽  
Wilco K Smits ◽  
Rogier C Buijsman ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Translational Research ◽  
Lymphoblastic Leukemia ◽  
Wild Type ◽  
Mtor Inhibition ◽  
Cytotoxic Effects ◽  
Downstream Signaling ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Equity Ownership

Abstract Background: Pediatric T-cell acute lymphoblastic leukemia patients frequently harbor mutations in IL7Ra or downstream molecules encoded by JAK1, JAK3, N-RAS, K-RAS, NF1, AKT, and PTEN. These mutated signaling molecules can contribute to leukemia by disturbing a multitude of cellular processes such as the cell cycle, epigenetics, apoptosis, or affecting other important signal transduction pathways. Aims: We aimed to determine the overall incidence of mutations in IL7Ra and downstream signaling components in a large cohort of pediatric T-ALL patients. In order to find better treatment options for patients with these mutations, we analyzed the effect of selected IL7Ra-pathway inhibitors-individually and in combinations-on downstream signaling and cytotoxicity in Ba/F3 cells expressing each of the mutations. Methods: We sequenced 146 pediatric T-ALL patient samples for mutations in the FERM, pseudokinase and kinase domains of the Janus kinase gene family (JAK1, JAK2, JAK3, TYK2) and hotspot regions of N-RAS and K-RAS. We adapted the IL3-dependent Ba/F3 cell line to express mutant or wild type genes upon induction by doxycycline and assessed cell viability and signaling in the absence of IL3. Various IL7Ra-pathway inhibitors were tested using this system, and the synergy of combined inhibitors was determined by comparing the dose-response curve of different ratios of IC50-based inhibitor concentrations to the curves for each of the single inhibitors. The Combination Index was calculated using Calcusyn™ software. Results: IL7Ra, JAK, RAS, AKT and PTEN mutations are present in approximately 45% of patients and occur in a predominantly mutually exclusive fashion, suggesting they share aberrant activation of similar downstream targets. We found JAK1, JAK3 and RAS mutations as previously reported, but also identified new JAK1 mutations including V427M, L624YPILKV, E668Q, P815S, and T901G. A novel three-dimensional model of JAK1 reveals that mutations in JAK molecules affect important amino acids that are involved in the interaction between the pseudokinase and kinase domains, facilitating constitutive kinase activity. In our doxycycline-inducible IL3-dependent Ba/F3 system, expression of mutant genes-in contrast to the wild type genes-transforms Ba/F3 cells by supporting IL3-independent growth through activation of the RAS-MEK-ERK and PI3K-AKT pathways. We used this system to test the sensitivity to pharmacological inhibitors; IL7Ra and JAK mutant Ba/F3 cells are sensitive to JAK inhibition, so JAK inhibitors such as ruxolitinib may offer therapeutic potential for IL7Ra, JAK1 or most JAK3 mutated T-ALL patients. The RAS and AKT mutants respond to RAS-MEK and PI3K-AKT-mTOR inhibition, respectively, but are-as expected-insensitive to JAK inhibition. Remarkably, IL7Ra and JAK mutants are relatively resistant to downstream RAS-MEK-ERK or PI3K-AKT-mTOR inhibition, indicating that inhibiting just one of these downstream pathways is insufficient. We provide evidence of (cross-)activation of the alternate pathway when one of these pathways is inhibited. Combined inhibition of MEK and PI3K/AKT synergistically prevents proliferation of the IL7Ra- and JAK-mutants by efficiently blocking both downstream signaling pathways. Furthermore, this combined inhibition is cytotoxic in two out of five tested primary T-ALL specimens. Summary/Conclusion: We show that the combined inhibition of MEK and PI3K/AKT leads to strong and synergistic cytotoxic effects in the IL7Ra and JAK mutants and efficiently blocks signaling downstream of both pathways. This inhibitor combination is effective in two out of five primary T-ALL samples. Therefore, the cytotoxic effects of synergistic MEK and PI3K/AKT inhibition should be further explored as a therapeutic option for (relapsed) ALL patients. Disclosures Buijsman: Netherlands Translational Research Center B.V.: Equity Ownership, Other: founder and shareholder. Zaman:Netherlands Translational Research Center B.V.: Equity Ownership, Other: founder and shareholder.

A novel PAX5-ELN fusion protein identified in B-cell acute lymphoblastic leukemia acts as a dominant negative on wild-type PAX5

Blood ◽  
2006 ◽  
Vol 109 (8) ◽  
pp. 3417-3423 ◽  
Author(s):  
Marina Bousquet ◽  
Cyril Broccardo ◽  
Cathy Quelen ◽  
Fabienne Meggetto ◽  
Emilienne Kuhlein ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Quantitative Polymerase Chain Reaction ◽  
Dominant Negative ◽  
Leukemic Cells ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Cell Acute Lymphoblastic Leukemia

Abstract We report a novel t(7;9)(q11;p13) translocation in 2 patients with B-cell acute lymphoblastic leukemia (B-ALL). By fluorescent in situ hybridization and 3′ rapid amplification of cDNA ends, we showed that the paired box domain of PAX5 was fused with the elastin (ELN) gene. After cloning the full-length cDNA of the chimeric gene, confocal microscopy of transfected NIH3T3 cells and Burkitt lymphoma cells (DG75) demonstrated that PAX5-ELN was localized in the nucleus. Chromatin immunoprecipitation clearly indicated that PAX5-ELN retained the capability to bind CD19 and BLK promoter sequences. To analyze the functions of the chimeric protein, HeLa cells were cotransfected with a luc-CD19 construct, pcDNA3-PAX5, and with increasing amounts of pcDNA3-PAX5-ELN. Thus, in vitro, PAX5-ELN was able to block CD19 transcription. Furthermore, real-time quantitative polymerase chain reaction (RQ-PCR) experiments showed that PAX5-ELN was able to affect the transcription of endogenous PAX5 target genes. Since PAX5 is essential for B-cell differentiation, this translocation may account for the blockage of leukemic cells at the pre–B-cell stage. The mechanism involved in this process appears to be, at least in part, through a dominant-negative effect of PAX5-ELN on the wild-type PAX5 in a setting ofPAX5 haploinsufficiency.

TYK2-STAT1 Pathway Positively Regulates BCL2 Gene Expression in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1470-1470
Author(s):  
Takaomi Sanda ◽  
Jeffrey W Tyner ◽  
Alejandro Gutierrez ◽  
Vu N Ngo ◽  
Jason M Glover ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Protein Expression ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Wild Type ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bcl2 Protein ◽  
Bcl2 Expression

Abstract Abstract 1470 To discover oncogenic pathways that are characteristically deregulated in T-cell acute lymphoblastic leukemia (T-ALL), we performed RNA interference screens both in T-ALL cell lines and primary specimens. We found that the JAK tyrosine kinase family member, TYK2, and its downstream effector, STAT1, are each required for the survival of T-ALL cells. To identify the effector molecules downstream of the TYK2-STAT1 pathway in T-ALL, we analyzed global gene expression profiles in TYK2-dependent T-ALL cell lines after silencing of TYK2 or STAT1. As expected, gene set enrichment analysis revealed that genes downregulated by TYK2 knockdown were generally also downregulated by knockdown of STAT1. Importantly, we found that expression of the anti-apoptotic gene BCL2 was significantly downregulated after silencing of both TYK2 and STAT1. Analysis by quantitative PCR of additional T-ALL cell lines revealed that silencing of TYK2 resulted in significant reductions of BCL2 mRNA expression in multiple TYK2-dependent cell lines. Expression of the wild-type but not the kinase-dead TYK2 protein was sufficient to rescue BCL2 protein expression and to prevent apoptosis after knockdown of endogenous TYK2, indicating that the tyrosine kinase activity of TYK2 is required for BCL2 upregulation. Similarly, expression of the shRNA-resistant wild-type STAT1A protein partially rescued BCL2 protein expression and prevented apoptosis, while a variant of STAT1A (Y701F) that is incapable of becoming phosphorylated on a requisite tyrosine residue did not rescue BCL2 levels. Taken together, our findings indicate that aberrant activation of a TYK2-STAT1 pathway upregulates BCL2 expression in T-ALL cells, and that the T-ALL cells develop pathway dependence, in that they require these sustained high levels BCL2 expression for survival. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Protein Translocation Inhibitors Overcome Cytokine-Induced Glucocorticoid Resistance in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 805-805
Author(s):  
Lauren K. Meyer ◽  
Cristina Delgado-Martin ◽  
Phillip P. Sharp ◽  
Dustin McMinn ◽  
Christopher J. Kirk ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Surface ◽  
Research Funding ◽  
Secretory Pathway ◽  
Signal Sequence ◽  
Lymphoblastic Leukemia ◽  
Life Sciences ◽  
Employment Equity ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Equity Ownership

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.

scholarly journals Frequency of IKZF1 Deletions in a Peruvian Population with B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 11-11
Author(s):  
Daniel J Enriquez ◽  
Rachel J. Mitchell ◽  
Krisztina Zuborne Alapi ◽  
Elizabeth Cervantes ◽  
Karina Cancino ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Dominant Negative ◽  
White Blood Count ◽  
Induction Treatment ◽  
Wild Type ◽  
Cell Acute Lymphoblastic Leukemia

Frequency of IKZF1 Deletions in a Peruvian Population with B-cell Acute Lymphoblastic Leukemia Background: B-cell Acute Lymphoblastic Leukemia (B-ALL) is an aggressive disease with worse outcomes in older patients, and latino ethnicity. Additionally, Latino populations are at higher risk of developing B-ALL.IKZF1is an essential lymphoid transcription factor with deletions (ΔIKZF1)implicated in treatment failure and relapses. We aimed to evaluate the frequency ofIKZF1deletions in a cohort of Peruvian patients with newly diagnosed B-ALL. Methods: We collected diagnostic bone marrow samples from 41 consecutive patients with B-ALL diagnosed between 2015-2019 at Instituto Nacional de Enfermedades Neoplasicas (INEN; Lima, Peru). Bone marrow samples were cryopreserved prior to induction treatment. DNA was extracted using High Pure PCR Template Preparation Kit (Roche) at INEN. Samples with adequate DNA were screened forΔIKZF1by multiplex endpoint PCR covering four main deletions - dominant negative Δ4-7 or the loss of function Δ2-7, Δ4-8, and Δ2-8 IKZF1 deletions at UCL Cancer Insitute (London, UK) using the primers described by Caye et. al. We analyzed outcomes byIKZF1status. Results: Forty-one cases were enrolled during the study period. Clinical characteristics are presented in Table 1. Median age was 20 years[1-63]. Fifteen∆IKZF1cases (37%) were detected (67%BCR-ABL1 negand 33%BCR-ABL1pos).Cases withΔIKZF1were older than those with wild-typeIKZF1(median age 31 vs 13 years, p=0.002). Median presenting white blood count (WBC) was 48 x109/L [R:2-218], with a higher WBC inΔIKZF1compared to wild-type (87 vs 24 x109/L, p=0.001). The most frequent deletion was ∆4-7 (sevenBCR-ABL1 negand threeBCR-ABL1 pos) additional deletions are described in table 2. All patients received intensive 'pediatric-based' treatment, 21 with BFM-2009 and 19 with the CALGB 10403 protocol. CR rates after induction were 67% and 92% for∆IKZF1and wild-type cases, respectively. Eleven (73%) of patients with∆IKZF1subjects (73%) were MRD positive by flow cytometry after induction compared to 11 (44%) among wild-type. At a median follow-up of 2 years EFS was 38% in the∆IKZF1group and 58% in the wild type group, correspond OS was 38% and 58%, respectively. Conclusion: A high frequency of IKZF1 deletions was found in a Peruvian population with B-ALL and was associated with older age and higher presenting white blood counts. Prospective studies with larger Latino population are warranted to confirm this finding. Disclosures No relevant conflicts of interest to declare.

The MDM2 Inhibitor NVP-CGM097 Is Highly Active in a Randomized Preclinical Trial of B-Cell Acute Lymphoblastic Leukemia Patient Derived Xenografts

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 797-797 ◽  
Author(s):  
Elizabeth C Townsend ◽  
Tiffany DeSouza ◽  
Mark A. Murakami ◽  
Joan Montero ◽  
Kristen Stevenson ◽  
...  
Keyword(s):  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Wild Type ◽  
Employment Equity ◽  
Highly Active ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Equity Ownership ◽  
Relapsed Disease ◽  
Mdm2 Inhibitor

Abstract The majority of drugs tested in clinical trials for hematologic malignancies ultimately fail to confer benefit. In many cases, this results from the inability to accurately identify patients most likely to benefit and mechanisms that mediate resistance. At the same time, clinical trials commonly fail to capture essential specimens at early and late timepoints that could be used to identify predictors of response, pharmacodynamic markers of on-target activity and targetable resistance pathways. To overcome these challenges, we have developed a repository of >200 patient-derived xenografts (PDXs) of leukemias and lymphomas now available through the DFCI Public Repository of Xenografts (PRoXe; http://www.proxe.org). Because PDX models can be developed and propagated from a large number of patients, randomized in vivo studies using adequate numbers of PDXs offer the first opportunity to capture the diversity of disease biology in pre-clinical drug testing. We tested the novel MDM2 inhibitor CGM097, which is currently undergoing clinical testing in solid tumors, in 24 B-cell acute lymphoblastic leukemia (B-ALL) PDXs (including hypodiploid, near haploid, MLL- rearranged, CRLF2-rearranged, and BCR-ABL models) in a randomized, phase II-like trial. Only a small subset of B-ALLs harbor de novo TP53 mutations, suggesting that MDM2 antagonists may have broad activity in this disease. Each PDX was injected into 4 NOD.SCID.IL2Rɣ-/- (NSG) mice. Upon engraftment (>2% hCD45+/hCD19+ cells in the peripheral blood), mice were randomized to vehicle or CGM097 treatment arms. One animal from each treatment arm was sacrificed 26 hours after beginning treatment to examine pharmacodynamic endpoints. The remaining two mice continued on daily therapy until moribund. CGM097 markedly improved overall survival (median 73 vs 28 days for vehicle; p=0.0008). All 19 models with survival benefit from CGM were TP53 wild-type. Among 6 models (all TP53 wild-type) derived from patients with relapsed disease, the median survival improvement compared to vehicle was 53 days (p=0.0059), consistent with robust single-agent activity in relapsed disease. Specimens at the 26 hour timepoint and upon progression to moribund were captured from the majority of mice in the trial, allowing for comprehensive characterization of the trial population. Dynamic BH3 profiling (Montero et al. Cell 2015), in which CGM097 or vehicle is added to leukemia cells harvested from mice and the effect of CGM097 on "priming" for apoptosis was performed on 10 models and demonstrated 100% accuracy in predicting response to CGM097. To characterize the effects of MDM2 inhibition on p53-dependent gene expression, we measured expression of 120 p53-related genes using a custom Nanostring gene expression panel. Differential expression analysis identified 11 genes that were significantly upregulated (p≤0.05) by CGM097 treatment at the 26 hour timepoint, including the canonical p53 targets BBC3, CDKN1A and MDM2. All mice treated with CGM097 ultimately became moribund from progressive leukemia. Targeted deep sequencing identified acquired TP53 mutations in only 2 leukemias after progression on CGM097. This indicates that p53 mutation is not the primary genetic driver of resistance to MDM2 inhibition in B-ALL PDXs. Despite this, CGM097-dependent transcriptional changes were largely abolished in the majority of leukemias collected from mice upon progression on CGM097. In summary, we established a paradigm for "Phase II-like" trials in panels of human leukemia PDX models. With this approach, we defined CGM097 as a highly active agent across the diverse spectrum of TP53-wildtype B-ALL, and established 19 independent models of acquired resistance that are the ideal reagents for defining mechanisms and then testing combinations in vivo that overcome those mechanisms. The same paradigm could be applied as a new standard for pre-clinical testing of drugs to minimize the empiric nature of current drug development strategies. Disclosures Barzaghi-Rinaudo: Novartis Institutes for Biomedical Research: Employment. Letai:AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding. Jeay:Novartis: Employment, Equity Ownership. Wuerthner:Novartis: Employment, Equity Ownership. Halilovic:Novartis: Employment, Equity Ownership.

scholarly journals Loss of PTEN in Pediatric T-Cell Acute Lymphoblastic Leukemia Patients: Proteomic and Molecular Characterization

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2408-2408
Author(s):  
Paola Bonaccorso ◽  
Cristina Bugarin ◽  
Chiara Buracchi ◽  
Grazia Fazio ◽  
Maddalena Paganin ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Protein Extraction ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Strong Association ◽  
Mtor Pathway ◽  
Wild Type ◽  
Pten Protein ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Signaling networks such as the PI3K/Akt/PTEN/mTOR pathway play a role in the modulation of the aggressiveness of T-Cell Acute Lymphoblastic Leukemia (T-ALL), but the significance of an aberrant activation of this pathway is poorly investigated. The master regulator of PI3K-AKT signaling is the lipid phosphatase and tensin homolog (PTEN); decreased or absent PTEN expression or activity could be associated with a constitutive activation of PI3K/Akt/PTEN/mTOR signaling pathway in T-ALLs. We investigated PTEN Exon7 mutations in diagnostic DNA samples from 67 pediatric T-ALL enrolled in AIEOP ALL2000 and R2006 protocols according to methods by Bandapalli et al. (Haematologica, 2012). We also investigated the PI3K/Akt/PTEN/mTOR pathway using Western Blotting (WB) and Flow Cytometry (FC) in parallel. WB analysis was performed using standard RIPA buffer for protein extraction. FC analysis of protein expression was applied as previously described (Gaipa G et al, Leukemia 2008), and protein levels were measured as % of positive cells compared to isotype control. Positivity or negativity by WB was established by presence or absence of a protein band, while for FC a threshold for positivity was set at ≥ 1% of positive cells. PTEN Exon 7 mutations were identified in 11 out of 67 (16.4%) of patients. According to samples availability, PI3K/Akt/PTEN/mTOR pathway was studied in 9 out of 67 patients (3 PTEN Exon 7 mutated and 6 wild type). PTEN protein resulted completely absent in all three PTEN Exon 7 mutated patients, by contrast PTEN was expressed in all 6 PTEN exon 7 wild type patients (mean by FC 46.98% ± 28.58%). This finding was fully confirmed when WB was applied to the same samples. We did not observe any statistically significant differences in p4EBP1 or mTOR levels in Exon 7 mutated patients as compared to wild type. By contrast, PTEN Exon 7 mutated blasts showed lower phospho-S6 levels compared to PTEN Exon7 wild type patients (mean 3.0% of positive cells vs 32.0%, p=ns). In conclusion, our data show a frequency of PTEN Exon 7 mutations of 16.4%, in agreement with the report by Bandapalli et al. (17.3%). Interestingly, we observed a strong association between the PTEN Exon 7 mutation and the total absence of PTEN protein in the pediatric T-ALL patients studied (data not reported so far to our knowledge). Moreover, although the differences are not statistically significant, p-S6 expression resulted consistently lower in mutated patients as compared to wild type patients. If confirmed in a larger cohort of pediatric T-ALLs, our data could open new insights in the significance of PTEN Exon 7 mutation in pediatric T-ALL and its associated functional profile. Disclosures No relevant conflicts of interest to declare.

Activating NOTCH1 Mutations Are Associated with Good Treatment Response in Childhood T-Cell Acute Lymphoblastic Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1444-1444
Author(s):  
Stephen Breit ◽  
Martin Stanulla ◽  
Thomas Flohr ◽  
Martin Schrappe ◽  
Wolf-Dieter Ludwig ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Confidence Interval ◽  
Treatment Response ◽  
Lymphoblastic Leukemia ◽  
Wild Type ◽  
Cell Acute Lymphoblastic Leukemia ◽  
The Impact ◽  
Notch1 Mutations

Abstract T-cell acute lymphoblastic leukemia (T-ALL) accounts for 10–15 % of pediatric ALL. Very rare cases of T-ALL (< 1 %) harbor the chromosomal translocation t(7;9) that involves NOTCH1, a gene encoding a single-pass, heterodimeric transmembrane receptor. NOTCH1 has an essential function in early intrathymic T-cell development. Recently, it has been demonstrated that more than 50 % of childhood T-ALLs carry activating mutations within the NOTCH1 gene (Weng et al., Science 2004). In the present study, we systematically analyzed the impact of activating NOTCH1 mutations on treatment response in 108 pediatric T-ALLs, registered in the ongoing ALL-BFM 2000 trial. In 56 cases (51.8%) activating NOTCH1 mutations were identified, located either in the heterodimerization (38/56 mutations; 65.5%), in the PEST (10/56; 17.9%) or in both domains (8/56; 14.3%). The presence of activating NOTCH1 mutations was significantly correlated with good prednisolone (p = 0.001, c2 or Fisher’s exact test) and MRD response (p = 0.002). T-ALLs with NOTCH1 mutations were 3.7 times more likely to show a good prednisolone response (95% confidence interval = 1.64–8.33; p = 0.002) and 4.8 times more likely to show a favorable MRD response (95% confidence interval = 2.04–11.11; p = 0.0003) when compared to patients with wild type NOTCH1. Patients with mutated NOTCH1 were thus underrepresented in the high risk group of the ALL-BFM 2000 protocol. This influence of NOTCH1 mutational status on risk stratification was independent from other commonly used criteria, like age and initial white blood cell count (WBC) at the time of diagnosis. Considering the impact of NOTCH1 mutations on long term prognosis, we analyzed those 49 patients of this cohort with a median follow-up of > 4 years. Eight patients relapsed within this follow-up period, 2 patients with mutated and 6 with wild type NOTCH1. With this small number of relapses, this trend towards a favorable influence of activating NOTCH1 mutations on EFS did not reach statistical significance. In conclusion, T-ALLs with NOTCH1 mutations are demonstrated to be more sensitive than those without to the ALL-BFM 2000 treatment strategy and may show a lower rate of relapse.

Identifying Drug-Resistant Mutations in Ebf1-Pdgfrb Ph-like Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1423-1423 ◽  
Author(s):  
Thai Hoa Tran ◽  
Jonathan Van Nguyen ◽  
Catherine C. Smith ◽  
Kathryn G. Roberts ◽  
Charles G. Mullighan ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Point Mutation ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Kinase Domain ◽  
Drug Resistant ◽  
Wild Type ◽  
Full Spectrum

Abstract Background: Advances in cancer genomics have recently identified a particular group of patients who display a gene expression profile (GEP) similar to that of Philadelphia-chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) patients in approximately 15% of children and over 25% of young adults with B-ALL; thus known as Ph-like ALL. The latter has a worse prognosis compared to those without the Ph-like GEP with conventional chemotherapy. Recent studies have unraveled the genomic landscape of Ph-like ALL which is characterized by genetic alterations activating kinase signaling pathways predicted to respond to tyrosine kinase inhibitors (TKIs). In light of the remarkable outcomes of Ph+ALL patients through incorporation of TKI, the Children's Oncology Group (COG) ALL Committee is actively working to incorporate dasatinib for Ph-like ALL patients harboring ABL-class kinase fusions (ABL1, ABL2, PDGFRB, CSF1R) and eventually, ruxolitinib for those with lesions that are predicted to respond to JAK inhibition. While it is hoped that many of these patients will be cured with the addition of relevant TKIs to chemotherapy, we hypothesize that a proportion of patients will develop resistance to TKI, similar to adults with chronic myeloid leukemia who have been treated with long-term TKI. Hence, investigating the underlying mechanisms governing therapy resistance in Ph-like ALL becomes critical for proactively developing novel therapeutic strategies in the relapsed setting. Objectives: To identify the full spectrum of mutations conferring resistance to clinically-active TKIs in Ph-like ALL and to characterize their relative biochemical resistance to different TKIs. Methods: We first focused on the EBF1-PDGFRB rearrangement since this is the most common recurrent kinase-activating fusion genes in pediatric Ph-like ALL. We used a previously validated in-vitro saturation mutagenesis screen to predict the full spectrum of EBF1-PDGFRB drug-resistant mutations. In brief, EBF1-PDGFRB plasmid was propagated into DNA-repair-deficient E. Coli strain XL-1 Red to generate a library of random mutants. Mutagenized EBF1-PDGFRB plasmid was transfected into 293T cells. Viral supernatants were collected and used to infect Ba/F3 cells. Transduced Ba/F3 cells were plated in 1.2% Bacto-agar and exposed to different TKIs (imatinib, dasatinib) at various concentrations. Genomic DNA from IL-3 independent and TKI-resistant colonies was isolated. The PDGFRB kinase domain was amplified and bidirectional sequencing was performed. Results: Our in-vitro screens showed that the vast majority of drug-resistant clones harbor a kinase domain (KD) point mutation. The predominant KD point mutation conferring resistance to imatinib (94%; 168/178 colonies) or dasatinib (81%; 338/416 colonies) was T681I, which is analogous to BCR-ABL1 T315I gatekeeper mutation. N666S was the second most common KD mutation (6%; 18/321 colonies). The full panel of KD mutations recovered is shown in Table 1. Ba/F3 cells harboring mutant EBF1-PDGFRB T681I was 100 times more resistant to dasatinib compared to wild-type and could be rescued by ponatinib, as predicted (Figure 1). Conclusion: Our screens suggest that KD point mutations may represent the primary mechanism of acquired TKI resistance in EBF1-PDGFRB Ph-like ALL. T681I was the most common KD point mutation in EBF1-PDGFRB upon exposure to imatinib or dasatinib. Future efforts should focus on targeting the T681I gatekeeper mutation with ponatinib or alternative agents for relapsed Ph-like ALL patients harboring these mutations. Figure 1. Cell proliferation profile of Ba/F3 cells harboring EBF1-PDGFRB wild-type and mutant T681I treated with dasatinib or ponatinib. Figure 1. Cell proliferation profile of Ba/F3 cells harboring EBF1-PDGFRB wild-type and mutant T681I treated with dasatinib or ponatinib. Figure 2. Figure 2. Disclosures Smith: Astellas: Research Funding; Plexxikon: Research Funding. Mullighan:Cancer Science Institute: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Speakers Bureau; Incyte: Consultancy, Honoraria; Loxo Oncology: Research Funding. Shah:Bristol-Myers Squibb: Research Funding; Pfizer: Research Funding; Plexxikon Inc.: Research Funding.

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