scholarly journals Pediatric Preclinical Testing Consortium Evaluation of the Novel Anti-Microtubule Drug E7130 in Xenograft Models of Early T-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3875-3875
Author(s):  
Richard B. Lock ◽  
Kathryn Evans ◽  
Connor D Jones ◽  
Narimanne El-Zein ◽  
Katerina Bendak ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Cell Precursor ◽  
Abcb1 Gene ◽  
Preclinical Testing ◽  
Qrt Pcr ◽  
Nsg Mice ◽  
Pediatric All ◽  
Pdx Models

Introduction: While children diagnosed with acute lymphoblastic leukemia (ALL) experience close to a 90% likelihood of cure, the outcome for certain high-risk pediatric ALL subtypes as well as adult ALL remains poor. Several standard-of-care drugs used in multi-agent treatment protocols for ALL (including vincristine, daunorubicin and methotrexate) are substrates for the ATP-dependent drug efflux pump P-glycoprotein (P-gp), encoded by the ABCB1 gene, although there are limited reports of ABCB1 gene expression being associated with poor outcome in ALL (Carrillo et al, Hematol. 22:286-91, 2017). A previously identified high-risk subtype of T-ALL (early T-cell precursor ALL, ETP-ALL) characterized by poor early response to conventional induction treatment, expresses significantly higher levels of the ABCB1 gene compared with typical T-ALL (1.97-fold; false discovery rate=0.0026; P=0.00029; Zhang et al, Nature 481:157-63, 2012). E7130 is a novel anti-microtubule drug that is a less potent substrate for P-gp compared with other anti-microtubule drugs such as vincristine, and it has shown significant preclinical activity against patient-derived xenograft (PDX) models of adult malignancies. Therefore, it was of interest for the Pediatric Preclinical Testing Consortium (PPTC) to test the in vivo activity of E7130 against its PDX models of pediatric ETP-ALL. Methods: ABCB1 mRNA expression in ALL PDXs was quantified by RNAseq (https://pedcbioportal.org) and qRT-PCR. P-gp protein expression was assessed by immunoblotting, while its activity was measured by the Rhodamine-123 efflux assay in the absence or presence of the P-gp inhibitor tariquidar. E7130 and vincristine were evaluated in vivo against 6 ETP-ALL PDXs. Each PDX was inoculated into 6-8 immune-deficient (NSG) mice per treatment group (2-5 x 106 cells/mouse). Engraftment and drug responses were evaluated by enumerating the proportion of human CD45+ cells in the peripheral blood (%huCD45+) at weekly intervals. E7130 was tested at 2 dose levels (0.09 and 0.135 mg/kg IV), while vincristine was evaluated at 1 mg/kg IP. Both drugs were administered weekly x 3. Events were defined as the %huCD45+ ≥25%. Drug efficacy was assessed by event-free survival of treated (T) and control (C) groups by T-C, T/C and stringent objective response criteria (Houghton et al, Pediatr Blood Cancer 49:928-40, 2007). Results: RNAseq analysis of the 6 ETP-ALL PDXs in the PPTC panel of 90 pediatric ALL PDXs revealed 3 with high ABCB1 expression (FPKM 7.1-13.6; ETP-2, -3 and -6) and 3 with low expression (FPKM 0-0.15; ETP-1, -4 and -5), which was confirmed by qRT-PCR and immunoblotting. Moreover, high levels of tariquidar-sensitive Rhodamine-123 efflux activity were confirmed in the 2 high ABCB1 expressing PDXs tested (ETP-2 and -3). E7130 was generally well tolerated in NSG mice, with maximum average weight loss of 2.7-17.6% in the groups treated with the highest dose compared with 3.2-12.7% in the vincristine treated groups. E7130 (0.09 mg/kg) significantly (P<0.05) delayed the progression of all 6 PDXs (T-C 10.5-41.3 days, T/C 1.8-5.9) and elicited objective responses in 2/6 PDXs (1 Complete Response, CR; 1 Maintained CR, MCR). The higher dose of E7130 (0.135 mg/kg) significantly delayed the progression of all 5 evaluable PDXs (T-C 18.1-49.5 days, T/C 2.3-8.8) and elicited 4 objective responses (1 CR, 3 MCRs). In contrast, vincristine significantly delayed the progress of 5/6 PDXs (T-C 3.5-37.7 days, T/C 1.3-6.6) and elicited 2 objective responses (1 CR, 1 MCR). When the PDXs were stratified around ABCB1 expression there was a trend for reduced vincristine activity against high (T-C 3.5-18.0 days, T/C 1.3-2.1) versus low (T-C 21.4-37.7 days, T/C 2.7-6.6) expressing PDXs. In contrast, the activity of E7130 was maintained regardless of ABCB1 expression at both the 0.09 mg/kg dose (low ABCB1, T-C 17.6-41.3 days, T/C 2.4-5.9; high ABCB1, T-C 10.5-32.0 days, T/C 1.8-3.0) and the 0.135 mg/kg dose (low ABCB1, T-C 30.7-46.2 days, T/C 3.5-8.8; high ABCB1, T-C 18.1-49.5 days, T/C 2.9-4.0). Conclusions: E7130 exhibits significant in vivo activity against pediatric ETP-ALL PDXs, regardless of their levels of ABCB1 expression. Our results support further evaluation of E7130 in pediatric ALL to determine whether it represents an alternative treatment option in ALL with high ABCB1 expression. (Supported by NCI Grants CA199000 and CA199922) Disclosures No relevant conflicts of interest to declare.

scholarly journals A Single Mouse Trial Platform for Evaluation of Novel Agents in Acute Lymphoblastic Leukemia By the Pediatric Preclinical Testing Consortium

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4010-4010
Author(s):  
Richard B. Lock ◽  
Kathryn Evans ◽  
Tara Pritchard ◽  
Cara Toscan ◽  
Chelsea Mayoh ◽  
...  
Keyword(s):  
Drug Testing ◽  
Lymphoblastic Leukemia ◽  
Objective Response ◽  
Novel Agents ◽  
Rna Seq ◽  
Single Experiment ◽  
Preclinical Testing ◽  
Nsg Mice ◽  
Pediatric All

Abstract Introduction: While children diagnosed with the most common pediatric malignancy, acute lymphoblastic leukemia (ALL), now experience close to a 90% likelihood of cure, the outcome for several high-risk subtypes remains poor. Furthermore, since standard-of-care drugs are extremely effective in this disease, and there are relatively few patients eligible for early phase clinical trials, only the most promising new agents are advanced for clinical evaluation following rigorous preclinical testing. However, conventional preclinical testing of novel agents is not sufficiently resourced to be able to encompass the vast heterogeneity of pediatric ALL, and new approaches to preclinical testing are required in this disease. The purpose of this study was to evaluate the utility of a single mouse trial (SMT) platform for preclinical assessment of novel agents on an almost clinical trial scale, to encompass the broad heterogeneity of pediatric ALL in a single experiment, and to identify molecular biomarkers associated with in vivo drug responses when carried out using molecularly-annotated patient-derived xenografts (PDXs). Methods: Eighty pediatric ALL PDXs broadly representative of all pediatric ALL subtypes were characterized in terms of engraftment kinetics in immune-deficient NSG mice, and molecularly annotated by RNA-seq, exome-seq and DNA copy number analysis. Between 2-5 million cells from each PDX were inoculated via the tail vein into 2 NSG mice/PDX. Starting at 2 weeks post inoculation engraftment was monitored by flow cytometric enumeration of the proportion of human CD45+cells in the murine peripheral blood (%huCD45+). When the %huCD45+ for each PDX reached >1% one mouse/PDX was treated with the established drug and topoisomerase I inhibitor topotecan (Tpt, 0.6 mg/kg IP daily x 5 x 2 weeks, repeated at 21 days) and the other mouse was treated with the experimental drug and second mitochondria-derived activator of caspases (SMAC)-mimetic birinapant (Bpt, 15 mg/kg IP every 3 days x 5). Treatment response was monitored using stringent objective response criteria modeled after the clinical setting, by mouse event-free survival (EFS where an event was defined as 25% huCD45+), and by waterfall plots comparing the maximum decrease in %huCD45+at any point post treatment initiation. The authenticity of each PDX was verified using a 60-allele SNP array both at the time of inoculation and at relapse post drug treatment for all mice. Results: Retrospective analysis of 1,000 samples of "single mouse" data previously showed that the single mouse results predicted the overall group response from conventional testing 75.3% of the time, which increased to 94.3% if a deviation of ± one response category was allowed. SMT results were achieved for 71 (88.8%) and 73 (91.3%) of the intended 80 mice for Tpt and Bpt, respectively. Waterfall plots revealed that 60/71 (84.5%) and 30/73 (41.1%) of PDXs achieved regressions in response to Tpt and Bpt treatment, respectively. When compared with historical objective response measures from conventional drug testing carried out by the Pediatric Preclinical Testing Consortium (PPTC) the SMT results showed high concordance for both Tpt (R=0.904; P=0.014; n=7 PDXs) and Bpt (R=0.804; P<0.0001; n=20 PDXs). Moreover, the SMT confirmed the preferential in vivo efficacy of Bpt against B-ALL compared with T-ALL, early T-cell precursor ALL (ETP-ALL) and ALL with mixed linage leukemia gene rearrangements (MLLr-ALL) that was previously revealed by conventional testing carried out by the PPTC. Analysis of divergent responses observed within the MLLr-ALL subpanel (n=9 PDXs) to Tpt revealed a potential 40 gene signature model, with up-regulated genes in Responders being associated with transcription regulation, cellular proliferation and differentiation. Conclusions: This study has shown that SMTs provide an accurate and cost-effective platform for preclinical drug testing in pediatric ALL on an almost clinical trial scale. Moreover, SMTs can almost encompass the heterogeneity of pediatric ALL in a single experiment, and are likely to be useful for large-scale correlations with in vitro drug sensitivity data. Finally, SMTs have the power to identify molecular biomarkers of in vivo response to established and novel drugs in pediatric ALL when combined with exome-seq, RNA-seq and DNA copy number analysis of molecularly-annotated PDXs. Supported by U01CA199000 from the NCI. Disclosures No relevant conflicts of interest to declare.

scholarly journals Opposing regulation of BIM and BCL2 controls glucocorticoid-induced apoptosis of pediatric acute lymphoblastic leukemia cells

Blood ◽  
2015 ◽  
Vol 125 (2) ◽  
pp. 273-283 ◽  
Author(s):  
Duohui Jing ◽  
Vivek A. Bhadri ◽  
Dominik Beck ◽  
Julie A. I. Thoms ◽  
Nurul A. Yakob ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Glucocorticoid Receptor ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Pediatric Acute Lymphoblastic Leukemia ◽  
Intronic Region ◽  
Key Points ◽  
Pediatric All ◽  
Induced Apoptosis

Key Points The glucocorticoid receptor coordinately regulates the antiapoptotic BCL2 and proapoptotic BIM genes in pediatric ALL cells in vivo. GR binding at a novel intronic region is associated with BIM transcription and dexamethasone sensitivity in pediatric ALL cells in vivo.

In vivo monitoring of JAK/STAT and PI3K/mTOR signal transduction inhibition in pediatric CRLF2-rearranged acute lymphoblastic leukemia (ALL).

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 9506-9506
Author(s):  
Sarah Kathleen Tasian ◽  
Shannon L. Maude ◽  
Junior Hall ◽  
Tiffaney Vincent ◽  
Charles Grenfell Mullighan ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Nsg Mice ◽  
Signal Transduction Inhibitors ◽  
Background Therapy ◽  
Pediatric All ◽  
Therapeutic Responses

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). Splenocytes were briefly stimulated ex vivo with thymic stromal lymphopoietin (ligand for CRLF2) and stained with human-specific surface and intracellular phosphoantibodies for multi-parameter phosphoflow cytometry analysis. Results: Ruxolitinib-induced inhibition of phospho (p)-JAK2 and pSTAT5 was most pronounced in non-CRLF2r ALLs with novel JAK2-activating BCR-JAK2 and IL7RA/LNK mutations. Sirolimus potently inhibited pS6 and other PI3K/mTOR pathway phosphoproteins in the CRLF2r r ALLs. PSTAT5 and pS6 inhibition correlated with longer-term ruxolitinib- and sirolimus-induced decreases in ALL cell burden, demonstrating therapeutic responses to STIs. Conclusions: Ruxolitinib inhibited JAK/STAT phosphosignaling and markedly decreased leukemic burden in the JAK2-activating BCR-JAK2 and IL7RA/LNK mutant ALL xenografts. Sirolimus potently inhibited PI3K/mTOR (as well as some JAK/STAT) phosphosignaling and had greater therapeutic efficacy than ruxolitinib in the CRLF2r ALLs. The safety of ruxolitinib and of temsirolimus with cytotoxic chemotherapy are currently being established in Children’s Oncology Group Phase I trials.

scholarly journals Evaluation of Patient-Derived Xenografts for Modeling Outcome of Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3759-3759
Author(s):  
Abdulmohsen M Alruwetei ◽  
Hernan Carol ◽  
Rosemary Sutton ◽  
Glenn M Marshall ◽  
Richard B Lock
Keyword(s):  
Gene Expression ◽  
Drug Resistance ◽  
Clinical Outcome ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Nsg Mice ◽  
Relapse Prediction ◽  
Pediatric All ◽  
Selection Of

Abstract Introduction: Children with acute lymphoblastic leukemia (ALL) are stratified at diagnosis based on molecular/cytogenetic characteristics and their response to initial treatment to receive risk-adapted multi-agent chemotherapy. The majority of ALL patients are stratified as Intermediate Risk (IR) and present with moderate levels of minimal residual disease (MRD<5x104) after receiving induction therapy, although an unacceptably high proportion of these patients relapse. The lack of specific prognostic features makes it difficult to predict the response of IR patients to treatment. The early identification of patients who are destined to relapse would facilitate improvements in tailored treatments for IR ALL patients. Recent progress in the development of patient-derived xenografts (PDXs) in immune-deficient mice represents an opportunity to improve relapse prediction in ALL patients. The aims of this study were to: (1) optimize the engraftment conditions of IR pediatric ALL samples to predict patient response to treatment; and, (2) to assess the development and mechanisms of therapy-induced drug resistance. Methods: Two pairs of IR pediatric ALL patients were matched based on clinical and genetic features, except that one patient from each pair relapsed early while the other remains relapse-free (ALL-Rel and ALL-CR1, respectively). Three parameters were varied in establishing PDXs by inoculating one million bone marrow (BM) derived biopsy cells collected at diagnosis into groups of 4 mice: (1) mouse strain (NOD/SCID vs. NSG); (2) site of inoculation (intravenous vs. intra-femoral); and (3) early treatment of mice with a 2-week induction chemotherapy regimen of vincristine, dexamethasone, and L-asparaginase (VXL). Leukemia engraftment was monitored weekly based on the proportion of human versus mouse CD45+ cells in the murine PB, and the median times to engraftment were compared according to patient outcome. The median time to engraft was also compared between the VXL-treated and non-treated groups. PDXs harvested from mice were compared for ex vivo sensitivity to single agent vincristine, dexamethasone and L-asparaginase. PDX gene expression profiles were also compared to identify pathways associated with evasion of VXL treatment in vivo. Results: The efficiency of engraftment was greater for NSG mice (29/32 mice engrafted) versus NOD/SCID mice (20/32 mice), and primary ALL cells also engrafted significantly faster in NSG mice (median time to engraft 71.1 days) compared with NOD/SCID mice (83.5 days) (P < 0.01), with no apparent difference associated with clinical outcome. Intrafemoral inoculation did not improve the efficiency or speed of engraftment compared with intravenous inoculation, nor predicted clinical outcome. However, PDX responses to VXL induction chemotherapy reflected the clinical outcome of the patients from whom they were derived; those derived from the 2 ALL-Rel patients exhibited in vivo drug resistance (leukemia growth delay of 1 and 6.2 days) compared with those derived from the 2 ALL-CR1 patients (34.7 and >119.8 days). Further, ex vivo analysis showed that the PDXs derived from the ALL-Rel patients exhibited resistance to vincristine or L-asparaginase compared with those derived from the ALL-CR1 cases. Moreover, the in vivo VXL treatment of an ALL-CR1 PDX resulted in selection of cells that exhibited vincristine resistance. Gene expression profiling revealed significant up-regulation of microtubule associated proteins (MAPs) and tubulin isotypes (alpha and beta) in vincristine-resistant PDXs. Genes that were significantly upregulted in vincristine resistant PDXs with a false discovery rate (FDR) < 0.05 and P value < 0.02 include TUBB6, TUBA1A, TUBA1B, MAP1S, TUBA3D and TBCA. The increased expression of genes that affect microtubule functions suggest that changes in microtubule dynamics and/or stability led to decreased sensitivity to antimicrotubule agents. Conclusions: In vivo selection of PDXs with an induction-type regimen of chemotherapeutic drugs may lead to improved relapse prediction and identify novel mechanisms of drug resistance in IR pediatric ALL. Support: Steven Walter Foundation; NHMRC Australia, APP1057746 Disclosures No relevant conflicts of interest to declare.

scholarly journals Selective Inhibition of Class II HDAC Isoforms 4 and 5 Provides a Promising Therapeutic Intervention for MLL-Rearranged Acute Lymphoblastic Leukemia in Infants

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2206-2206
Author(s):  
Tamara C.A.I. Verbeek ◽  
Susan Arentsen-Peters ◽  
Patricia Garrido Castro ◽  
Sandra Pinhancos ◽  
Kirsten Vrenken ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Hdac Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Hdac Inhibitors ◽  
Selective Inhibition ◽  
Class Ii ◽  
In Vivo Efficacy ◽  
Pdx Models

Abstract MLL-rearranged acute lymphoblastic leukemia (ALL) is characterized by deregulation of the epigenome and shows susceptibility towards epigenetic perturbators such as histone deacetylase (HDAC) inhibitors. Hence, HDACs represent attractive drug targets and a variety of small molecule HDAC inhibitors have been developed and evaluated for the treatment of hematological malignancies. However, most broad-spectrum inhibitors, which simultaneously target the majority of human HDAC isoforms, often induce toxicity, especially in combination with other therapeutic agents. Therefore, selective inhibition of only one or two HDAC isoforms may represent a better alternative, provided that disease-specific dependency on specific HDACs has been identified. We examined the effects of shRNA-mediated knock-down of the class II HDACs (i.e. HDAC4, HDAC5, HDAC6, HDAC7 and HDAC9) in the MLL-rearranged ALL cell lines SEM and ALL-PO. Except for HDAC9, loss of expression (both on the mRNA and protein level) of all HDACs led to strong reductions in viable cells (0.70 to 0.19-fold; p=0.02-0.0016) in both models due to apoptosis, cell cycle arrest, or a combination thereof. Next, we evaluated the in vitro efficacy of a variety of class II HDAC-specific inhibitors on a panel of MLL-rearranged ALL (n=5) using 4-day viability MTT assays. This revealed that the selective HDAC4/5 inhibitor LMK-235 was able to recapitulate the loss-of-function phenotype of HDAC4 and HDAC5. Dose response curves showed complete growth inhibition in MLL-rearranged ALL cell lines (n=5), as well as in primary MLL-rearranged infant ALL patient samples (n=4), with IC 50 values of ~100 nM and 40-100 nM, respectively. Importantly, at these concentrations, LMK-235 hardly affected whole bone marrow samples derived from healthy individuals (n=2), for which IC 50 values were ~1 µM. To further explore the potential of class II HDAC inhibitor-based therapeutic strategies, we performed a combinatorial drug screen to identify compounds that synergize with LMK-235. For this, a compound library (comprising &gt;200 unique agents) was screened in the absence and presence of varying concentrations of LMK235 in the MLL-rearranged cell line models SEM and ALL-PO. This, and subsequent validation experiments in additional cell line models, revealed that Venetoclax (BCL2 inhibitor), Trametinib (MEK/ERK inhibitor), Ponatinib (multi-tyrosine kinase inhibitor) and Omipalisib (a PI3K/mTOR inhibitor) strongly synergized with LMK-235. Average ZIP synergy scores ranged from 10-30, with peak ZIP scores up to 40. Importantly, synergistic effects were consistent over all concentration combinations tested. The addition of 50-100 nM LMK-235 strongly reduced IC 50 values for Omipalisib, Ponatinib and Venetoclax (0.27-fold p=0.003, 0.11-fold p=0.0005, 0.75-fold p=0.0004, respectively) in both models. In preparation to assess the in vivo efficacy of LMK-235 in patient-derived xenograft (PDX) mouse models of MLL-rearranged infant ALL, pharmacokinetic/pharmacodynamic (PK/PD) analysis was performed in immunodeficient NSG mice (n=5). For this, mice were treated with 20 mg/kg of LMK-235, daily administered via intraperitoneal injections for a total of 29 days. While none of the mice showed signs of toxicity or weight loss, LMK-235 plasma levels were stably maintained at concentrations that are highly effective against MLL-rearranged ALL cells in vitro. Taken together, these data demonstrate that various class II HDAC isoforms are targetable vulnerabilities in MLL-rearranged ALL and that pharmaceutical inhibition of HDAC4/5 by LMK-235 represents an attractive therapeutic option. Moreover, high levels of synergy observed between this HDAC inhibitor and various agents belonging to drug classes already reported to be effective against MLL-rearranged ALL, warrants pre-clinical evaluation in vivo. Currently, the assessment of the in vivo efficacy of LMK-235 monotherapy in MLL-rearranged infant ALL PDX models is in progress, after which promising synergistic HDAC inhibitor-based drug combinations will be evaluated. To determine the additional therapeutic value, the efficacy of LMK-235 and promising synergistic combinations will be evaluated in the background of conventional combination chemotherapy, where PDX models will receive a mouse-adapted version of induction therapy currently applied for treatment of MLL-rearranged infant ALL patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Identification of Potent BH3-Mimetic Combinations Targeting Pro-Survival Pathways in Human B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 567-567
Author(s):  
Donia M Moujalled ◽  
Diane T Hanna ◽  
Giovanna Pomilio ◽  
Veronique Litalien ◽  
Shaun Fleming ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Advisory Committee ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Lymphoblastic Leukemia ◽  
Bh3 Mimetics ◽  
Nsg Mice ◽  
Bh3 Mimetic

Abstract Background Precursor-B acute lymphoblastic leukemia (B-ALL) is an aggressive hematological malignancy. Relapsed disease has a poor prognosis, despite improved outcomes with tyrosine kinase inhibitors for Ph+ cases and immunotherapeutic approaches, such as blinotumomab and CAR-T cells. Targeting cell survival with novel small molecule BH3-mimetic inhibitors of BCL-2 (e.g. Souers et al Nat Med 2013, Roberts et al, NEJM 2016 and Casara et al, Oncotarget 2018), BCL-XL (Lessene et al, Nat Chem Biol, 2013) or MCL1 (Kotschy et al, Nature 2016) is an emerging therapeutic option. BCL-2 is reported to have a pro-survival role in BCR-ABL1, JAK2 fusion, ETV6-RUNX1 and MLL-r driven ALL (Brown et al., Journal Biological Chemistry 2017). BH3-mimetics targeting BCL-2 and BCL-XL has efficacy in paediatric ALL xenografts (Khaw et al., Blood 2016), while ruxolitinib combined with ABT-737 is synergistic in JAK2-mutant pre-B-ALL (Waibel et al., Cell Reports 2013). We now report that combined targeting of BCL-2 and MCL1 has broad pre-clinical efficacy in adult B-ALL samples with Ph+, Ph- and Ph-like characteristics. Methods S55746 and S63845 were obtained from Servier/Novartis, A1331852 from Guillaume Lessene (WEHI), venetoclax, daunorubicin, dexamethasone (DXM) and tyrosine kinase inhibitors (TKIs) from Selleckchem. Bliss synergy scores were determined using a checkerboard approach to evaluate combinations (previously described Bliss, Ann Appl Biol 1939). Primary ALL cells were obtained from 14 patients (4 Ph+ and 10 Ph-) providing informed consent. Ex vivo cell viability (sytox blue exclusion) at 48h was determined over a 5-log dilution range (1nM-10uM) using drugs alone or in equimolar combinations. For in vivo studies, adult B-ALL patient derived xenografts were performed in NSG; NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Results Dual BH3-mimetic targeting of BCL-2 and MCL1 was strongly synergistic (Bliss sum >1000) in SUPB15 (Ph+ BCR-ABL1), BV173 (lymphoid blast crisis BCR-ABL1), MUTZ5 (Ph-like) and MHHCALL4 (Ph-like) B-ALL cell lines. This was more effective than single BH3-mimetic combinations with DXM or TKIs (dasatinib or ruxolitinib) (Fig. A, B). In B-ALL patient samples, combined BCL-2 and MCL1 targeting lowered the LC50 by 10-1000 fold (to LC50<10nM) in 4/4 Ph+ ALL cases and 8/10 Ph- cases. Similarly, combined MCL1 and BCL-XL targeting demonstrated synergy in 3/4 Ph+ cases and 7/10 Ph- cases (to LC50<10nM), confirming remarkable anti-leukemic activity compared to BH3-mimetics alone or chemotherapy (daunorubicin) (Fig. C). BH3-mimetic combination therapy (S55746/S63845) compared favourably in Ph+ ALL cases to S55746 (figure D) or S63845 (Figure E) in combination with dasatinib. Preliminary data using patient-derived xenografts in NSG mice revealed in vivo efficacy of combined S55746 and S63845 therapy against 3 adult B-ALL cases (1 Ph+ and 2 Ph-). Reduction of established ALL in the bone marrow was observed in mice receiving combined S55746/S63845 after one week of treatment (p=<0.05) (Fig. F-H). Conclusions Dual BH3-mimetic targeting of BCL-2 and MCL1 induces synergistic killing of human B-ALL cell lines and primary ALL samples in vitro and rapid cytoreduction in vivo. Simultaneous inhibition of BCL-2 and MCL1 represents a novel and effective approach for targeting Ph+, Ph- and Ph-like B-ALL without need for additional DNA-damaging chemotherapy or kinase inhibition. Our results support the translational investigation of dual BH3-mimetic targeting of BCL-2 and MCL1 in the clinic. Figure legend: BLISS synergy scores for A. Ph+ and B. Ph-like ALL cell lines for drug combinations targeting BCL-2, MCL1, BCR-ABL, JAK1/2 and DXM. C. LC50 activity in primary ALL after 48hr of treatment with BH3 mimetics and combinations targeting BCL-2, MCL1, BCL-XL, compared to daunorubicin (LC50< 10nM red; ~ 100nM yellow; >1uM green). D. Comparison of BH3-mimetics targeting D.BCL-2 or E. MCL1 in combination with dasatinib in Ph+ vs Ph- primary B-ALL samples. Activity expressed as LC50 activity after 48h, with median values shown. Irradiated NSG mice were transplanted with 106 primary B-ALL cells. Engraftment of F. Ph+ and G-H. Ph- B-ALL cells was confirmed at 10 weeks by detection of hCD45 in PB. Mice were then treated with i) vehicle (d1-5), ii) S55746 100mg/kg days 1-5 by gavage, iii) S63845 25 mg/kg IV on days 2 and 4 or iv) S55746+S63845. Mice were euthanized on day 8 and hCD45+ from flushed femurs quantified. Disclosures Chanrion: Servier: Employment. Maragno:servier: Employment. Kraus-Berthier:servier: Employment. Lessene:servier: Research Funding. Roberts:Janssen: Research Funding; AbbVie: Research Funding; Genentech: Research Funding; Walter and Eliza Hall: Employment, Patents & Royalties: Employee of Walter and Eliza Hall Institute of Medical Research which receives milestone and royalty payments related to venetoclax. Geneste:servier: Employment. Wei:Pfizer: Honoraria, Other: Advisory committee; Celgene: Honoraria, Other: Advisory committee, Research Funding; Amgen: Honoraria, Other: Advisory committee, Research Funding; Servier: Consultancy, Honoraria, Other: Advisory committee, Research Funding; Novartis: Honoraria, Other: Advisory committee, Research Funding, Speakers Bureau; Abbvie: Honoraria, Other: Advisory board, Research Funding, Speakers Bureau.

scholarly journals Targeted Cancer Therapy in High-Risk Pediatric Leukemia Using Global Phosphotyrosine Profiling

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 969-969
Author(s):  
Sibasish Dolai ◽  
Keith CS Sia ◽  
Alissa K Robbins ◽  
Ling Zhong ◽  
Sue Heatley ◽  
...  
Keyword(s):  
High Risk ◽  
B Cell ◽  
Tyrosine Phosphorylation ◽  
Lymphoblastic Leukemia ◽  
Cell Precursor ◽  
Pediatric Leukemia ◽  
Pediatric All ◽  
Quantitative Tool

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

scholarly journals Low Epression of MiR-18a Distinguishes Pediatric and Adult Acute Lymphoblastic Leukemia from Each Other

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4991-4991
Author(s):  
Neda Mosakhani ◽  
Mohamed El Missiry ◽  
Emmi Vakkila ◽  
Päivi Heikkilä ◽  
Sakari Knuutila ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Immune Cell ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Tissue Samples ◽  
Inflammatory Microenvironment ◽  
Qrt Pcr ◽  
Response To Chemotherapy ◽  
Pediatric All

Abstract In several adult solid cancers the presence or absence of an inflammatory microenvironment has turned out to be an important prognostic factor. Acute lymphoblastic leukemia (ALL) is seen in both adults and children but the response to chemotherapy and survival is significantly worse in adults than children. Therefore, we wanted to study whether the expression of immune system associated molecular markers would be different in adult and pediatric ALL patients at the time of diagnosis. IDO and FOXP3 were studied from paraffin embedded tissue samples by immunohistochemistry in 12 pediatric and 10 adult bone marrow samples. Inflammation associated miRNA analysis were performed in 19 adult and 79 pediatric ALL patients and involved miR-10, miR-15, miR-16, miR-17-92 cluster, miR-33, miR-146a, miR-150, miR-155, miR-181a, miR-222, miR-223, and miR-339. miRNAs were first analysed by Agilent's miRNA microarray and thereafter validated by qRT-PCR. miRNAs not expressed in at least 75% of one group of samples were excluded. Significance (p <0.05; q<0.1) of differential expression was estimated by t-test for those miRNAs with at least a 2.0 fold change. Sufficient RNA for qRT-PCR was available for 42 pediatric and 19 adult patients. The adult and pediatric ALL patients had quantitatively and qualitatively similar expression of IDO and FOXP3 in leukemic bone marrow samples (p=0.26 and 0.74, respectively). Out of studied miRNAs only miR-18a differed significantly in microarray analysis between adult and pediatric ALL being lower in children (FC -3.74; p 0,0037). Results were confirmed by qRT-PCR (upregulated in adults, FC 3.71, p 0.003161). The other members of the miR-17-92 cluster did not differ significantly. We conclude that pediatric and adult ALL patients have remarkably similar pattern of immune cell associated markers in bone marrow at diagnosis. This is in line with recent evidence that the outcome of the adult ALL patients can be significantly improved if treated with pediatric protocols. However, the low expression of miR-18a in pediatric ALL is interesting and demands further studies. Disclosures No relevant conflicts of interest to declare.

Drug Screening of Primary Human Pediatric Pre-B Cell Acute Lymphoblastic Leukemia (pre-B ALL) Cells in Their Stromal Niche

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4295-4295
Author(s):  
Jae-Hung Shieh ◽  
Tsann-Long Su ◽  
Jason Shieh ◽  
Malcolm A.S. Moore
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Culture System ◽  
Lymphoblastic Leukemia ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 4295 Pre-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common leukemia in children and is treatable. However, no in vitro nor in vivo models are available to investigate their pathophysiology other than a number of established cell lines that grow in the absence of any cytokine dependence or stromal interaction. We developed a serum-free MS-5 cell (a murine bone marrow stromal cell line) co-culture system that is capable of expanding human primary pre-B ALL CD34+CD19+ cells in vitro. To define a population of pre-B ALL initiating cells, our study reveals that a sorted CD34bright population displays a slow proliferation and maintains a high % of CD34+ cells. In contrast, CD34dim cells/CD34− cells fraction shows a higher proliferation but expanded cells lost CD34 antigens. A group of alkylating molecules (BO-1055, -1090, 1099, -1393 and -1509) was evaluated for proliferation of the pre-B ALL CD34+ cells, the pre-B ALL CD34− cells, human mesenchymal stem cells (hMSC), murine MSC (MS-5 cells and Op9 cells), human bone marrow derived endothelial cells (BMEC), and human cord blood (CB) CD34+ cells, as well as for a week 5 cobblestones area forming (CAFC) assay with CB CD34+ cells. BO-1055 molecule efficiently suppressed the growth of pre-B ALL CD34+ cells (IC50 = 0.29 μM) and CD34− cells (IC50 = 0.31 μM). In contrast, IC50 of BMEC, MSC, CB CD34+ cells and CAFC are >10, >25, 8, and >5 μM, respectively. Pre-B ALL cells expressing green fluorescent protein (GFP) and luciferase (GFP-Lu-pre-B ALL) were created, and a xenograft of the GFP-Lu-pre-B ALL cells to NOD/SCID IL2R gamma null (NSG) mice was established. The in vivo effect of BO-1055 to the GFP-Lu-pre-B ALL cells in NSG mice is under investigation. Our stromal culture system supports primary pre-B ALL cells and closely recapitulates the growth of primary human pre-B ALL cells in their niche in vivo. Based on this co-culture system, we identified BO-1055 as a potential therapeutic agent with an excellent toxicity window between pre-B ALL cells and normal tissues including BMEC, MSC and hematopoietic progenitor/stem cells. The in vitro stromal co-culture system combined with the xenograft model of GFP-Lu-pre-B ALL cells provides an efficient and powerful method to screen new drugs for pre-B ALL therapy. Disclosures: No relevant conflicts of interest to declare.

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