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

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.

Download Full-text

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

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.9506 ◽

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.

Download Full-text

Epigenetic Silencing of the Pro-Apoptotic Bim Gene in Glucocorticoid Poor-Responsive Pediatric Acute Lymphoblastic Leukemia, and Its Reversal by Histone Deacetylase Inhibition.

Blood ◽

10.1182/blood.v114.22.939.939 ◽

2009 ◽

Vol 114 (22) ◽

pp. 939-939 ◽

Cited By ~ 1

Author(s):

Richard B Lock ◽

Petra S Bachmann ◽

Rocco G Piazza ◽

Carwyn Davies ◽

Mary James ◽

...

Keyword(s):

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Poor Response ◽

Dexamethasone Treatment ◽

Pediatric Acute Lymphoblastic Leukemia ◽

Dexamethasone Resistance ◽

Pediatric All ◽

Blast Count ◽

H3k9 Acetylation

Abstract Abstract 939 Introduction: The glucocorticoids dexamethasone and prednisolone are critical components of combination chemotherapy regimens used to treat pediatric acute lymphoblastic leukemia (ALL). While approximately 80% of patients are cured, poor response to upfront prednisolone monotherapy in 5-10% of patients is a strong predictor of adverse treatment outcome. A greater understanding of the mechanisms responsible for glucocorticoid resistance in pediatric ALL is likely to result in the design of novel strategies to overcome resistance and improve outcome for patients with refractory disease. The pro-apoptotic BH3-only BCL-2 family member BIM (BCL-2L11) has previously been identified as a critical component of glucocorticoid-induced apoptosis in normal and malignant lymphocytes. The purpose of this study was to elucidate clinically relevant mechanisms of glucocorticoid resistance in pediatric ALL, and design and test resistance reversal strategies. Patients and Methods: The study included biopsy specimens obtained at diagnosis from a cohort of patients who received 7 days of prednisolone monotherapy (60 mg/m⋀2/day) and a single age-related dose of intrathecal methotrexate, and whose prednisolone response was determined on Day 8 to be Good (PGR, peripheral blast count < 1 × 10⋀9/L, n=11) or Poor (PPR, peripheral blast count >= 1 × 10⋀9/L, n=11). The study also included biopsies from a cohort of patients who experienced early relapse (within 2 years of diagnosis, n=12). Experimental models of the disease included xenografts (n=12) established in immune-deficient (NOD/SCID) mice using direct explants of patient biopsies. Methods of analysis included: real-time quantitative RT-PCR and immunoblotting of glucocorticoid-induced mRNA and proteins following dexamethasone treatment of ex vivo cultured xenograft cells; DNA methylation analysis of the BIM 5' Untranslated Region (5'UTR) by methylated DNA immunoprecipitation (MeDIP), bisulfite sequencing, and SEQUENOM MassArray Epityper analysis; real time and array based chromatin immunoprecipitation (ChIP) analysis of histone-H3K9 acetylation, H3K4 and H3K27 tri-methylation across the entire BIM locus; as well as assessment of the histone deacetylase inhibitor SAHA (Vorinostat) to reverse dexamethasone resistance both in vitro and in vivo. Results: Dexamethasone resistance in pediatric ALL xenografts was consistently associated with failure to up-regulate BIM mRNA and protein in response to dexamethasone treatment, despite verification that other known glucocorticoid-responsive genes (GILZ, FKBP5) were highly induced in all xenografts. These results indicate specific silencing of BIM in dexamethasone-resistant xenografts rather than a dysfunctional glucocorticoid receptor, leading us to focus on epigenetic regulation of BIM transcription. DNA methylation of the BIM 5'UTR was variable between xenografts, and showed no clear association with dexamethasone resistance. In contrast, the extent of H3K9 acetylation at the BIM locus significantly correlated with the ability of dexamethasone to up-regulate BIM expression in ALL xenografts (R=0.90; P<0.001; n=12): relatively deacetylated H3K9 was associated with BIM repression, indicating that the BIM locus was in a “closed” and transcriptionally inaccessible conformation in dexamethasone-resistant xenografts. These results were in direct contrast to the other BH3-only genes examined, NOXA and PUMA, where H3K9 deacetylation was not associated with dexamethasone resistance. Moreover, H3K9 acetylation at the BIM promoter was significantly decreased in patients classified as PPRs compared with PGRs (P=0.013), while the decrease in BIM H3K9 acetylation in patients at early relapse approached a significant difference from PGRs (P=0.096). These results confirm that the results obtained with xenografts were not an artifact of engraftment. Treatment of a dexamethasone-resistant xenograft with SAHA increased BIM H3K9 acetylation, up-regulated BIM, and caused synergistic anti-leukemic efficacy with dexamethasone both in ex vivo cultures and in an in vivo model of systemic disease. Conclusions: Poor response to glucocorticoid therapy in pediatric ALL is significantly associated with epigenetic silencing of the BIM gene locus, and strategies aimed at improving glucocorticoid sensitivity and clinical outcome should consider incorporating epigenetic modifiers. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Leukemia Stem Cells in Acute Lymphoblastic Leukemia: Unveiling Hierarchical Structure at Single Cell Resolution

Blood ◽

10.1182/blood.v124.21.4786.4786 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4786-4786

Author(s):

Noriko Satake ◽

Connie Duong ◽

Sakiko Yoshida ◽

Ryan Davis ◽

Stephenie Liu ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

B Cell ◽

Single Cell ◽

Lymphoblastic Leukemia ◽

Cell Type ◽

Transcriptome Profile ◽

Nsg Mice ◽

Novel Method

Abstract Leukemia stem cells (LSCs) are the root of leukemia, and are responsible for drug resistance and disease relapse. However, LSCs have not yet been identified for acute lymphoblastic leukemia (ALL). Among many challenges, lack of phenotypic markers is one of the major problems in identifying ALL LSCs. In this study, we demonstrated a novel method to isolate LSCs from both T- and B- cell ALLs and further characterized their transcriptome profile at the single cell level. We have recently identified a novel method to isolate ALL LSCs based on cellular metabolic activities. We demonstrated that these isolated LSCs had in vivo leukemia-initiating capability (LIC). We have developed a series of primary ALL xenograft mouse models using patient samples and NOD/SCID/IL2Rg-/- (NSG) mice. Leukemia cells harvested from several generations of these mice were used in this study. We isolated LSCs and non-LSCs from 4 different B-cell type ALL samples and transplanted them separately into healthy NSG mice. Cell numbers used varied between 5, 10, and 50,000 per mouse, and the number of the animals varied between three and eight per group. All the animals transplanted with LSCs developed leukemia between 5-14 weeks, whereas those transplanted with non-LSCs did not develop the disease within the same timeframe or by the end of the study, which was more than 4 months after leukemia development in the LSC group. In order to characterize and identify potential therapeutic targets in the LSCs, we investigated the transcriptome profile of these cells. First, we performed genomewide microarray gene expression profiling of RNA isolated from the LSCs and non-LSCs using 4 ALL cell lines (Reh, JM1, Jurkat, and Molt4). There were 173 genes which showed at least 2-fold difference in gene expression between the LSCs and non-LSCs. Using a panel of primer sets for the 100 genes exhibiting the highest difference in expression, we performed qRT-PCR for these genes in the isolated LSCs and non-LSCs from 11 primary ALL samples (10 B-cell and 1 T-cell type) transplanted and harvested from our NSG xenograft mouse models at different generations. There was a distinct difference in the transcriptome profile between the LSCs and non-LSCs in these primary ALL samples. Overall gene expression of 93 LSC signature genes was much lower in the LSCs than in the non-LSCs. Recent advances in microfluidic technologies allowed us to investigate cells at single cell resolution. Growing evidence suggests that cancer stem cells consist of heterogeneous cell populations (subclones). Therefore, we further investigated whether these isolated LSCs have subclones using the Fluidigm C1 and Biomark system. Preliminary results using a primary ALL sample harvested from our xenograft mouse model, indicate that there are at least two distinct subclones in the LSCs based on principal component analysis of the single cell data. In summary, we 1) developed a novel method to isolate ALL LSCs which have in vivo LIC, 2) demonstrated that isolated LSCs have a distinct transcriptome profile, and 3) discovered that the LSCs seem to consist of subclones. Currently we are in the process of performing detailed comprehensive transcriptome analyses and additional single cell transcriptome assays. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Rearranged Infant Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v112.11.1913.1913 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1913-1913 ◽

Cited By ~ 1

Author(s):

Ronald W. Stam ◽

Monique L. Den Boer ◽

Pauline Schneider ◽

Jasper de Boer ◽

Jill Hagelstein ◽

...

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

Expression Profiles ◽

Lymphoblastic Leukemia ◽

Gene Expression Profiles ◽

Glucocorticoid Resistance ◽

Glucocorticoid Treatment ◽

Pediatric All

Abstract MLL rearranged Acute Lymphoblastic Leukemia (ALL) represents an unfavorable and difficult to treat type of leukemia that often is highly resistant to glucocorticoids like prednisone and dexamethasone. As the response to prednisone largely determines the clinical outcome of pediatric ALL patients, overcoming resistance to these drugs may be an important step towards improved prognosis. Here we compared gene expression profiles between prednisone-resistant and prednisone-sensitive pediatric ALL patients to obtain gene expression signatures associated with prednisone resistance for both childhood (>1 year of age) and MLL rearranged infant (<1 year of age) ALL. Merging both signatures in search for overlapping genes associated with prednisone resistance in both patient groups we, found that elevated expression of MCL-1 (an anti-apoptotic member of the BCL-2 protein family) appeared to be characteristic for both prednisone-resistant ALL samples. To validate this observation, we determined MCL-1 expression using quantitative RT-PCR in a cohort of MLL rearranged infant ALL samples (n=23), and confirm that high-level MCL-1 expression significantly confers glucocorticoid resistance both in vitro and in vivo. Finally, down-regulation of MCL-1 in prednisone resistant MLL rearranged ALL cells by RNA interference (RNAi) markedly sensitized these cells to prednisone. Therefore we conclude that MCL-1 plays an important role in glucocorticoid resistance and that MCL- 1 suppressing agents co-administered during glucocorticoid treatment may be beneficial especially for MLL rearranged infant ALL patients.

Download Full-text

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

Blood ◽

10.1182/blood-2018-99-113207 ◽

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.

Download Full-text

TTT-3002 Is Active Against Relapsed/Refractory AML Patient Bone Marrow With FLT3 TKI-Resistance Mutations At Residue D835

Blood ◽

10.1182/blood.v122.21.3818.3818 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3818-3818

Author(s):

Hayley S Ma ◽

Bao Nguyen ◽

Mark Levis ◽

Allen B Williams ◽

Donald Small

Keyword(s):

Drug Resistance ◽

Clinical Trials ◽

Ex Vivo ◽

Point Mutations ◽

Site Directed Mutagenesis ◽

Resistance Mutations ◽

Patient Sample ◽

Tki Resistance ◽

Selection Of

Abstract Over 35% of acute myeloid leukemia (AML) patients harbor a constitutively activating mutation in FLT3, either internal tandem duplication (ITD) or point mutations (FLT3/PM). FLT3/ITD mutations in particular confer a poor prognosis, and thus several FLT3 tyrosine kinase inhibitors (TKIs) have been developed and are currently in clinical trials. However, many have failed due to insufficient achievement of FLT3 kinase inhibition in vivo. This is thought to be due to insufficient potency, reduced activity against FLT3/PM, and/or selection for resistance-conferring point mutations in FLT3/ITD. Therefore, the search for novel FLT3 TKIs that overcome some of these resistance mechanisms that result in persistent FLT3 activation is necessary to improve the cure rate for this disease. We have previously reported on TTT-3002, a novel TKI that possesses the most potent activity against FLT3 phosphorylation, with IC50s of 100-250pM. Here we characterize the activity of this compound against TKI-resistant AML. The phenomenon of drug resistance associated with selection of FLT3 point mutations is being observed with increasing frequency as higher levels of inhibition by FLT3 TKIs are achieved in clinical trials of FLT3/ITD AML patients. In order to predict the ability of TTT-3002 to successfully treat some of these resistance mutations, we used site-directed mutagenesis to generate a series of FLT3/ITD TKI resistant cell lines. We directly compared the activity of TTT-3002 to other FLT3 TKIs currently in clinical trials including CEP-701, sorafenib, AC220 and PKC412. TTT-3002 remained the most potent inhibitor of all six FLT3/ITD TKI resistance mutants screened, with viability IC50 values of less than 1nM (with the single exception of Ba/F3-G697R/ITD, IC50=11nM). It was thus able to effectively inhibit FLT3 with TKI resistance mutations against which other TKIs are ineffective. Therefore, this compound’s broad spectrum of activity against FLT3/ITD TKI resistance mutations may enable it to successfully treat FLT3/ITD AML patients who have become resistant to other FLT3 TKIs. To examine this directly, human AML samples were obtained from patients that developed resistance to sorafenib or AC220 while on clinical trials and presented with a dual D835/ITD mutation or D835 point mutation alone at the time of relapse. These blasts were evaluated for their ex vivo sensitivity to TTT-3002, sorafenib and AC220, with a diagnostic FLT3/ITD+ patient sample for comparison. Encouragingly, we observed significant effects on proliferation, induction of apoptosis, and cell signaling when cells were treated with TTT-3002. AC220 was moderately active against FLT3/ITD+ blasts and one patient sample with a D835/ITD mutation, and sorafenib was active against only the diagnostic FLT3/ITD sample, but none of the three relapse samples. Therefore, TTT-3002 maintains activity against human AML patient samples that are resistant in clinical trials to the FLT3 TKIs sorafenib and AC220. To account for the patterns of drug resistance observed for different FLT3 TKIs in our study, we modeled the positions of staurosporine-like (TTT-3002, CEP-701 and PKC412) and sorafenib-like inhibitors (sorafenib and AC220) bound to FLT3. Examination of amino-acid substitutions in FLT3 known to confer resistance to TKIs from clinical trials shows that substituting arginine for glycine at position 697 likely leads to steric clashes with FLT3 TKIs. It has been proposed that the F691 residue forms a stabilizing π-π stacking interaction with AC220, and thus a mutation to a Leu accounts for reduced binding affinity for this TKI. TTT-3002 is predicted to bind FLT3 without making direct contact with the F691 residue, and thus is unaffected by mutations at this site accounting for its continued potency. The closer proximity of F691 and N676 to sorafenib compared to staurosporine likely underlies the trend for substitutions at these sites to confer resistance to sorafenib-like but not staurosporine-like inhibitors. In summary, TTT-3002 has the potential for increased clinical applicability due to its ability to target not only FLT3/ITD and FLT3/PM AML patients but also patients who have developed resistance to other FLT3 TKIs through the selection of resistance mutations within the FLT3/ITD allele. These preclinical studies further support the entry of TTT-3002 into human clinical trials. Disclosures: Levis: Ambit: Consultancy.

Download Full-text

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

Blood ◽

10.1182/blood-2019-123867 ◽

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.

Download Full-text

Integrin α6 mediates the drug resistance of acute lymphoblastic B-cell leukemia

Blood ◽

10.1182/blood.2019001417 ◽

2020 ◽

Vol 136 (2) ◽

pp. 210-223 ◽

Cited By ~ 2

Author(s):

Eun Ji Gang ◽

Hye Na Kim ◽

Yao-Te Hsieh ◽

Yongsheng Ruan ◽

Heather A. Ogana ◽

...

Keyword(s):

Drug Resistance ◽

Tyrosine Kinase ◽

B Cell ◽

Kinase Inhibitor ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Underlying Mechanism ◽

Conditional Knockout

Abstract Resistance to multimodal chemotherapy continues to limit the prognosis of acute lymphoblastic leukemia (ALL). This occurs in part through a process called adhesion-mediated drug resistance, which depends on ALL cell adhesion to the stroma through adhesion molecules, including integrins. Integrin α6 has been implicated in minimal residual disease in ALL and in the migration of ALL cells to the central nervous system. However, it has not been evaluated in the context of chemotherapeutic resistance. Here, we show that the anti-human α6-blocking Ab P5G10 induces apoptosis in primary ALL cells in vitro and sensitizes primary ALL cells to chemotherapy or tyrosine kinase inhibition in vitro and in vivo. We further analyzed the underlying mechanism of α6-associated apoptosis using a conditional knockout model of α6 in murine BCR-ABL1+ B-cell ALL cells and showed that α6-deficient ALL cells underwent apoptosis. In vivo deletion of α6 in combination with tyrosine kinase inhibitor (TKI) treatment was more effective in eradicating ALL than treatment with a TKI (nilotinib) alone. Proteomic analysis revealed that α6 deletion in murine ALL was associated with changes in Src signaling, including the upregulation of phosphorylated Lyn (pTyr507) and Fyn (pTyr530). Thus, our data support α6 as a novel therapeutic target for ALL.

Download Full-text

CBP/β-Catenin/FOXM1 Is a Novel Therapeutic Target in Triple Negative Breast Cancer

Cancers ◽

10.3390/cancers10120525 ◽

2018 ◽

Vol 10 (12) ◽

pp. 525 ◽

Cited By ~ 9

Author(s):

Alexander Ring ◽

Cu Nguyen ◽

Goar Smbatyan ◽

Debu Tripathy ◽

Min Yu ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Drug Resistance ◽

Triple Negative Breast Cancer ◽

Therapeutic Target ◽

Triple Negative ◽

Creb Binding Protein ◽

Novel Therapeutic

Background: Triple negative breast cancers (TNBCs) are an aggressive BC subtype, characterized by high rates of drug resistance and a high proportion of cancer stem cells (CSC). CSCs are thought to be responsible for tumor initiation and drug resistance. cAMP-response element-binding (CREB) binding protein (CREBBP or CBP) has been implicated in CSC biology and may provide a novel therapeutic target in TNBC. Methods: RNA Seq pre- and post treatment with the CBP-binding small molecule ICG-001 was used to characterize CBP-driven gene expression in TNBC cells. In vitro and in vivo TNBC models were used to determine the therapeutic effect of CBP inhibition via ICG-001. Tissue microarrays (TMAs) were used to investigate the potential of CBP and associated proteins as biomarkers in TNBC. Results: The CBP/ß-catenin/FOXM1 transcriptional complex drives gene expression in TNBC and is associated with increased CSC numbers, drug resistance and poor survival outcome. Targeting of CBP/β-catenin/FOXM1 with ICG-001 eliminated CSCs and sensitized TNBC tumors to chemotherapy. Immunohistochemistry of TMAs demonstrated a significant correlation between FOXM1 expression and TNBC subtype. Conclusion: CBP/β-catenin/FOXM1 transcriptional activity plays an important role in TNBC drug resistance and CSC phenotype. CBP/β-catenin/FOXM1 provides a molecular target for precision therapy in triple negative breast cancer and could form a rationale for potential clinical trials.

Download Full-text

Healthy versus inflamed lung environments differentially effect MSCs

European Respiratory Journal ◽

10.1183/13993003.04149-2020 ◽

2021 ◽

pp. 2004149

Author(s):

Sara Rolandsson Enes ◽

Thomas H. Hampton ◽

Jayita Barua ◽

David H. McKenna ◽

Claudia C. dos Santos ◽

...

Keyword(s):

Gene Expression ◽

Healthy Volunteers ◽

Cell Injury ◽

Ex Vivo ◽

Lavage Fluid ◽

Cell Therapies ◽

Inflammatory Microenvironment ◽

Self Recognition ◽

Clinical Investigations

BackgroundDespite increased interest in MSC-based cell therapies for the acute respiratory distress syndrome (ARDS), clinical investigations have not yet been successful and understanding of the potential in vivo mechanisms of MSC actions in ARDS remain limited. ARDS is driven by an acute severe innate immune dysregulation, often characterised by inflammation, coagulation, and cell injury. How this inflammatory microenvironment influences MSC functions remains to be determined.AimTo comparatively assess how the inflammatory environment present in ARDS lungs versus the lung environment present in healthy volunteers alters MSC behaviors.MethodsClinical grade human bone marrow-derived MSCs (hMSCs) were exposed to bronchoalveolar lavage fluid (BALF) samples obtained from ARDS patients or from healthy volunteers. Following exposure, hMSCs and their conditioned media were evaluated for a broad panel of relevant properties including viability, levels of expression of inflammatory cytokines, gene expression, cell surface HLA expression, and activation of coagulation and complement pathways.ResultsPro-inflammatory, pro-coagulant, and major histocompatibility complex (self recognition) related gene expression was markedly up-regulated in hMSCs exposed ex vivo to BALF obtained from healthy volunteers. In contrast, these changes were less apparent and often opposite in hMSCs exposed to ARDS BALF samples.ConclusionThese data provide new insights into how hMSCs behave in healthy versus inflamed lung environments strongly suggesting that the inflamed environment in ARDS induces hMSC responses potentially benefical for cell survival and actions. This further highlights the need to understand how different disease environments affect hMSC functions.

Download Full-text