Identification and in vitro validation of neoantigens for immune activation against high-risk pediatric leukemia cells

Abstract Abstract 2654 Poster Board II-630 Introduction: With the institution of multidrug, multiphase chemotherapy regimens, major improvements in clinical outcomes have been made in pediatric acute leukemia patients in the last thirty years. However, there remains a substantial percentage of pediatric patients who relapse and die of their disease, particularly with high risk ALL, T cell ALL and AML. It is possible that these patients' disease initiates from a leukemic stem cell such as those found in adult myeloid disease, or at the very least, harbor a chemo-resistant population. Our research has two main aims: first to evaluate the functional and phenotypic heterogeneity within standard risk (SR), high risk (HR) and relapsed (RD) pediatric leukemia. Second, to evaluate current treatment regimens for the selection of a chemo-resistant or LSC populations and then attempt to target this population with novel treatments. Methods: In vitro studies for functional heterogeneity include colony-forming assays (CFU) using methylcellulose and limiting-dilution suspension culture studies. Phenotypic heterogeneity is evaluated with multi-color flow cytometry and detection of alterations in aldehyde dehydrogenase activity. Xenograft studies in immune deficient mice are used to evaluate self-renewal capability, serial engraftment kinetics, and alterations in phenotype. Drug studies are performed by evaluating the differences in phenotype and CFU over time when treating with conventional induction chemotherapy or novel agents. Results: We have evaluated several SR and HR ALL samples in addition to some RD samples, which are paired with HR diagnostic samples. In vitro studies revealed the SR samples had little to no colony forming ability (0-1%) while the HR samples had approximately 3-5% and the RD samples 8-10% colony-forming ability. Likewise, the SR samples failed to engraft NOD-SCID mice while the HR samples, from patients with infantile ALL and the MLL translocation or T cell ALL, had robust engraftment in primary and secondary recipients. The engraftment kinetics were uniformly faster in secondary recipients. These findings suggest that HR leukemia may be the result of a leukemia-initiating cell with stem cell-like characteristics while SR ALL may arise from a more committed lymphoid progenitor. Interestingly, in the RD samples, several of the phenotypic markers are similar to that of the primary sample after treatment with induction therapy, particularly with regards to percentages of CD 34, 133-1, 133-2 and aldehyde dehydrogenase levels. Several HR samples have been exposed to induction chemotherapy (Decadron, Cytarabine, Doxorubicin and Vincristine), and the CFU potential and phenotype evaluated over a two-week time course. Notably, the majority of bulk disease is effectively killed, the CFU content actually increases two to three-fold, when an equivalent number of viable cells are analyzed. Furthermore, the phenotype reveals brighter staining with several proposed stem cell markers (CD34, 117, 133-1, 133-2, 123, and measurement of aldehyde dehyrogenase). These data indicate the selection of a chemo-resistant or LSC population. Conclusions: Our results to this point suggest important differences both functionally and phenotypically, between SR, HR and RD pediatric leukemia. These findings are consistent with what would be expected given clinical differences in each of these disease states and begins to establish a means of identifying a LSC or chemo-resistant population, which can be targeted with novel treatment regimens. Likewise, these techniques may also provide a means of evaluating for minimal residual disease (MRD) in a LSC or chemo-resistant population by identifying that population's phenotype by passaging the initial sample through serial murine engraftments or in vitro drug studies. Disclosures: No relevant conflicts of interest to declare.

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Preclinical Evaluation of Oncolytic Reovirus in Targeted Therapeutics for High-Risk Pediatric Leukemia

Blood ◽

10.1182/blood.v120.21.3571.3571 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3571-3571

Author(s):

Matthew F. Clarkson ◽

Aru Narendran ◽

Randal N. Johnston

Keyword(s):

Cell Death ◽

High Risk ◽

Cell Viability ◽

Cell Lines ◽

Leukemia Cell ◽

Treatment Strategies ◽

Leukemia Cells ◽

Western Blots ◽

Pediatric Leukemia ◽

Leukemia Cell Lines

Abstract Abstract 3571 Purpose: Leukemia is the most common malignancy in children. Improved treatment strategies in recent decades have yielded substantially enhanced outcomes for children with leukemia, reaching survival rates >80%. However, there remain significant issues with current treatment. Certain subgroups of patients who are resistant to or relapse from current treatments have a dismal prognosis. Furthermore, there are significant late effects of intensive treatments, including secondary cancers, neurocognitive defects, cardiotoxicity, obesity and infertility. For these reasons, novel treatment strategies are urgently needed for high-risk leukemia in children. Reovirus type 3 Dearing is a wild-type double-stranded RNA virus that has shown great promise as a selective oncolytic agent by its ability to replicate in transformed cells but not in normal cells. Although a number of early phase clinical studies have been completed in patients with advanced, refractory solid tumors in adults, systematic evaluation of this agent in the treatment of refractory pediatric leukemia has not been reported. As an initial step towards developing an oncolytics based treatment approach, we report preclinical data with respect to the activity, target validation, target modulation and drug combinability of reovirus in childhood leukemia cells. Experimental Design: A panel of pediatric leukemia cell lines representing high-risk molecular features such as Bcr-Abl, MLL rearranged and mixed lineage was used (n =6). Expression of JAM-A, the cell surface receptor for reovirus, was assessed by flow cytometry. The Ras Activation Assay Kit (EMD Millipore) was used to assess activity of the RAS protein. Western Blots were used to assess the activation (phosphorylation) of the signaling partners downstream of RAS. Cells treated with reovirus, chemotherapy drugs, or both for distinct treatment schedules were assessed for cell viability by the CellTiter-Glo© Luminescent Cell Viability Assay (Promega), and cell death by apoptosis was confirmed by cleavage of PARP. Productive viral infection was assessed by measuring reoviral protein synthesis by Western Blots, and reoviral replication was assessed by virus plaque titration assay. Drug synergies were calculated according to the method of Chou and Talalay. Results: Target validation assays showed the expression of JAM-A, which facilitates effective viral entry into malignant cells, in five of six cell lines. These cell lines also demonstrated differential activation of RAS and downstream kinases, suggesting targeted susceptibility of these cells to reovirus oncolysis. To further test this, we infected cells with reovirus for 1–4 days and assessed cytopathic effects. Using phase contrast microscopy, we observed the virus treated cell lines to demonstrate morphological changes characteristic of cell death following infection. Cell viability assays were used to quantify this effect, and the mechanism of cell death was determined to be apoptotic as evidenced by caspase-dependent cleavage of PARP. Reovirus-induced cell death was correlated with viral protein production and replication. Next, we screened for the ability of reovirus to induce synergistic activity in a panel of conventional and novel targeted therapeutic agents. Our studies showed that, in contrast to the current antileukemic agents, the Bcl-2 inhibitor BH3 mimetic ABT-737 was able to significantly synergize with reovirus in all cell lines tested. Conclusions: In our in vitro studies, oncolytic reovirus as a single agent showed potent oncolytic activity against all pediatric leukemia cell lines tested that express the receptor for reovirus, regardless of the status of the RAS signaling pathway. Further, we found reovirus-induced oncolysis can be enhanced by combination with Bcl-2 inhibition but was unaltered or antagonized by the other drugs indicating a key relationship between the two pathways. As such, our data for the first time, show that pediatric leukemia cells carry the potential to be targeted by reovirus induced oncolysis and the identification of drug synergy and the biomarkers of target modulation provide the basis for further studies to develop this novel therapeutic approach for clinical studies in the near future. Disclosures: No relevant conflicts of interest to declare.

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In vitro profiling of the sensitivity of pediatric leukemia cells to tipifarnib: identification of T-cell ALL and FAB M5 AML as the most sensitive subsets

Blood ◽

10.1182/blood-2005-04-1640 ◽

2005 ◽

Vol 106 (10) ◽

pp. 3532-3537 ◽

Cited By ~ 19

Author(s):

B. F. Goemans

Keyword(s):

T Cell ◽

Leukemia Cells ◽

Pediatric Leukemia

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Matched Targeted Therapy for Pediatric Patients with Relapsed, Refractory or High-Risk Leukemias: A Report from the LEAP Consortium

Blood ◽

10.1182/blood-2018-99-116948 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 261-261 ◽

Cited By ~ 1

Author(s):

Yana Pikman ◽

Sarah K. Tasian ◽

Maria Luisa Sulis ◽

Todd M Cooper ◽

Melinda Pauly ◽

...

Keyword(s):

Targeted Therapy ◽

High Risk ◽

Research Funding ◽

Drug Testing ◽

Genetic Alterations ◽

Tumor Board ◽

Leukemia Cells ◽

Advisory Committees ◽

Tier 1

Abstract Despite the remarkable pace of characterizing the genomics of pediatric acute leukemias, the integration of real-time sequencing results into clinical practice has lagged. With increased availability of molecularly-targeted therapies, the promise of matching genetic lesions in patients' leukemia cells to treatment has not yet been fully realized. We established the first pediatric leukemia clinical genomics consortium in the United States, known as the Leukemia Precision-based Therapy (LEAP) Consortium, which includes 13 major pediatric cancer institutions. We hypothesized that it is feasible to identify and match, in real-time, actionable alterations with a targeted therapy for pediatric patients with relapsed, refractory or high-risk leukemias or myelodysplastic syndrome (MDS). Using a combination of a DNA-based next-generation sequencing panel and RNA-based gene fusion testing, followed by data review by our multidisciplinary molecular tumor board, we are conducting a clinical trial to test this hypothesis. To date, we have enrolled and reviewed data from 143 patients stratified by disease status: Cohort 1, patients with relapsed or refractory leukemias (n=93), and Cohort 2, patients with de novo high-risk leukemias or MDS (n=50). A matched targeted therapy (MTT) recommendation has been made for 72% (n=103) of patients, tiered based on the level of evidence linking the mutation to potential activity of targeted therapy in the context of each patient's disease (Tier 1: 11%, Tier 2: 4%, Tier 3: 41%, Tier 4: 6%, Tier 5: 10%). Of the 44 patients in Cohort 1 with clinical follow-up data, 5 (11%) had alterations in therapy made based upon sequencing results and MTT recommendation. These include the use of the MEK inhibitor trametinib for RAS mutant acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) (n=2), dasatinib for B-ALL and T-ALL with NUP214-ABL1 translocations (n=2) and ponatinib for B-ALL with an ABL1 T315I mutation (n=1). In addition, this genomic data refined diagnosis and supported additional germline assessment in a subset of patients. In parallel to the genomic sequencing, we are conducting high-throughput drug sensitivity (HTS) assays to study in vitro anti-leukemia effects of a panel of up to 120 targeted inhibitors in the context of leukemia-associated genetic alterations. Of the initial 106 accrued patients, 40 (38%) had adequate amounts of blood or bone marrow for in vitro drug testing. All samples were tested in the inhibitor panel assay, and 65% of the samples yielded interpretable results. Inhibitor screening data was compiled for all 12 patients with genetic alterations resulting in Tier 1, 2 or 3 MTT recommendations. This subset of patients had leukemias with two distinct molecular profiles: 1) oncogenic RAS signaling pathway mutations or 2) oncogenic tyrosine kinase alterations. Our molecular tumor board recommended trametinib for the first group of patients and tyrosine kinase inhibitors (TKIs), specific to the mutation, for the second group of patients. In vitro HTS data analysis demonstrated dose-response sensitivity of leukemia cells with RAS pathway mutations to trametinib, many of which had half-maximal inhibitory concentration (IC50) less than 50 nM. Similarly, leukemia cells from patients with FLT3, ABL1 or KIT mutations generally showed dose-responses to relevant TKIs with low IC50s. Overall, HTS data were concordant with MTT recommendations informed by the sequencing results. This first in the US multi-institutional prospective leukemia genomics trial brings state-of-the-art clinical genetic testing, assessment of prognostic biomarkers, selection of patients for germline testing, and targeted therapeutic treatment recommendations to children and young adults with high-risk, relapsed or refractory leukemias or MDS. Our collaboration provides a unique opportunity to perform sophisticated patient-specific in vitro drug testing assays to impact MTT discovery efforts, which we plan to validate in vivo using patient-derived xenograft models where feasible. We believe that a model like the LEAP Consortium has the potential to transform precision medicine approaches for children with high-risk leukemias and to inform future genomics-guided therapeutic trials and drug discovery efforts. Disclosures Tasian: Aleta Biopharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding. Burke:JAZZ: Speakers Bureau; Shire: Speakers Bureau; AMGEN: Speakers Bureau. Tyner:AstraZeneca: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Janssen: Research Funding; Array: Research Funding; Constellation: Research Funding; Gilead: Research Funding; Aptose: Research Funding; Incyte: Research Funding; Genentech: Research Funding.

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

Blood ◽

10.1182/blood.v124.21.969.969 ◽

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.

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In Vitro Growth Inhibition, Target Modulation and Drug Synergy In Pediatric Leukemia By The Novel Proteasome Inhibitor Carfilzomib

Blood ◽

10.1182/blood.v122.21.2673.2673 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2673-2673

Author(s):

Aarthi Jayanthan ◽

Yibing Ruan ◽

Meaghan Hagerty ◽

Ravi Shah ◽

Tony Truong ◽

...

Keyword(s):

Proteasome Inhibitor ◽

Proteasome Inhibitors ◽

Leukemia Cells ◽

Toxicity Profile ◽

Pediatric Leukemia ◽

Over Expression ◽

Drug Synergy ◽

Gm Csf ◽

Induction Of Apoptosis

Abstract Introduction Despite the considerable progress that has been made in past two decades, relapsed or refractory leukemia still remains a leading cause of death in children. Acute and long term toxicities prevent significant further intensification of current chemotherapeutic regimens. Hence, effective pre-clinical data on new agents and novel therapeutic approaches are urgently needed. Recent studies have established proteasome inhibition as a distinctive and effective way to induce cytotoxicity in tumor cells that have acquired resistance to conventional chemotherapy. Although they represent a major advancement in the treatment of various hematological malignancies, the first generation proteasome inhibitors exhibit measurable off-target toxicities and the eventual development of resistance. Carfilzomib (CFZ) is a selective proteasome inhibitor that is structurally distinct from bortezomib and has shown efficacy and favorable toxicity profile in multiple myeloma patients. Mechanistically, carfilzomib has been shown to irreversibly bind and inhibit the chymotrypsin-like activity of the 20S proteasome and to cause the accumulation of polyubiquinated proteins resulting in cell cycle arrest, apoptosis, and suppression of tumor growth. Methods A diverse panel of pediatric leukemia derived cell lines and primary leukemia specimens (n = 12) were used to evaluate CFZ induced in vitro cytotoxicity using the Alamar blue assay. These cell lines include leukemia with abnormal FLT3 (FLT3-ITD and over-expression), Bcr-Abl fusion, extremely high white blood cell count variants, mixed lineage leukemia and Juvenile Myelomonocytic Leukemia (JMML) cells that are hyper-stimulated with exogenous GM-CSF. A luminescent based technique that individually measures protease activities associated with the proteasome complex in cultured cells (Promega, Cell-Based Proteasome-Glo Assay) was used to evaluate the mechanism of CFZ activity in these cells. Drug combination studies were carried out with etoposide, cytarabine, sorafenib and mefloquine using Chou and Talalay methodology. Target modulation, induction of apoptosis and the modulation of autophagy were evaluated by Western Blot analysis of cells treated with CFZ at defined time periods. Results Carfilzomib induced effective cytotoxicity in all leukemia cells tested (IC50 mean = 7 nM, range = 0.2 – 10 nM). Cell based proteasome assays confirmed the targeted and specific activity of CFZ in these cells. Infant leukemia cells with FLT3 over-expression were highly sensitive to CFZ followed by cells with ITD. Primary JMML cells that showed high growth stimulation with GM-CSF were also significantly affected by CFZ (IC50 = 0.2 nM). Although the extent of drug synergy varied between AML and ALL cells, CFZ synergized with all four agents (Combination Index (CI) mean = 0.47, range = 0.2 – 0.9). Induction of apoptosis by CFZ was evidenced by the increase in the active fragments of caspase 7 and 8 and PARP cleavage. CFZ also modulated autophagy by showing concentration regulated changes in p62 and LC3B. However, this effect appears to be restricted to AML cells. In vitro clonogenic assays using normal human CD34+ cells showed that even at 10 nM concentration, CFZ has no detectable inhibition on erythroid or myeloid colony formation. Discussion Carfilzomib is a potent, selective and irreversible inhibitor of the ubiquitin-proteasome pathway in cancer cells and has shown an acceptable toxicity profile in adult clinical trials. Our current data substantiates its potential as an active anti-leukemic agent in currently difficult to cure pediatric leukemia subtypes. Furthermore, we provide evidence on useful drug combinations and target modulation data to characterize the molecular mechanisms and biological correlates of distinct proteasome inhibitors in pediatric leukemia. This information provides key primary data for further in vivo studies and to design effective early phase clinical trials in the near future. Disclosures: No relevant conflicts of interest to declare.

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Growth arrest and induction of apoptosis in high-risk leukemia cells by strawberry components in vitro

Journal of Functional Foods ◽

10.1016/j.jff.2009.01.004 ◽

2009 ◽

Vol 1 (2) ◽

pp. 153-160 ◽

Cited By ~ 8

Author(s):

Susan J. Zunino ◽

David H. Storms ◽

Yanjun Zhang ◽

Navindra P. Seeram

Keyword(s):

High Risk ◽

Growth Arrest ◽

Leukemia Cells ◽

Induction Of Apoptosis

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Ginsenoside Rh2 Mitigates Pediatric Leukemia Through Suppression of Bcl-2 in Leukemia Cells

Cellular Physiology and Biochemistry ◽

10.1159/000430383 ◽

2015 ◽

Vol 37 (2) ◽

pp. 641-650 ◽

Cited By ~ 11

Author(s):

Xiaoru Wang ◽

Yulin Wang

Keyword(s):

Cell Lines ◽

Marrow Cell ◽

Leukemia Cell ◽

Leukemia Cells ◽

Luciferase Reporter ◽

Therapeutic Effects ◽

Ginsenoside Rh2 ◽

Pediatric Leukemia ◽

Leukemia Cell Lines

Background/Aims: Acute myeloid leukemia (AML) is a severe malignant cancer worldwide, in both adult and pediatric patients. Since bone marrow cell transplantation is seriously limited by the availability of the immune-paired donor sources, the therapy for pediatric leukemia remains challenging. Ginsenoside Rh2 (GRh2) is a well-characterized component in red ginseng, and has established therapeutic effects for different diseases, although whether GRh2 may have a therapeutic effect on pediatric leukemia has not been investigated. Methods: We examined the effects of GRh2 on the survival of mice in an acute leukemia model. We analyzed the effects of GRh2 on the cell viability of leukemia cell lines in vitro, using a CCK-8 assay and an MTT assay. We analyzed the effects of GRh2 on the apoptosis of leukemia cell lines in vitro, by flow cytometry. We analyzed the levels of Bcl-2 and microRNA-21 (miR-21) in GRh2-treated leukemia cells. Prediction of binding between miR-21 and 3'-UTR of Bcl-2 mRNA was performed by a bioinformatics algorithm and confirmed by a dual luciferase reporter assay. Results: GRh2 significantly prolonged the survival of mice with pediatric leukemia. GRh2 significantly decreased the viability of leukemia cells in vitro, through induction of apoptosis. GRh2 significantly decreased the levels of an anti-apoptotic protein Bcl-2 in leukemia cells, possibly through induction of miR-21, which suppressed the translation of Bcl-2 mRNA via 3'-UTR binding. Conclusion: GRh2 may be an effective treatment for pediatric leukemia, and GRh2 may induce apoptosis of leukemia cells through miR-21-modulated suppression of Bcl-2.

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Inhibition of PI3K/mTOR Pathways with GDC-0980 in Pediatric Leukemia: Impact on Abnormal FLT-3 Activity and Cooperation with Intracellular Signaling Targets

Current Cancer Drug Targets ◽

10.2174/1568009619666190326120833 ◽

2019 ◽

Vol 19 (10) ◽

pp. 828-837 ◽

Cited By ~ 1

Author(s):

Abdulhameed Al-Ghabkari ◽

Maneka A. Perinpanayagam ◽

Aru Narendran

Keyword(s):

Intracellular Signaling ◽

Leukemia Cell ◽

Mek Inhibitor ◽

Leukemia Cells ◽

Parp Cleavage ◽

Synergistic Activity ◽

Pediatric Leukemia ◽

Flt3 Inhibitor ◽

Molecule Inhibitor

Background:GDC-0980 is a selective small molecule inhibitor of class I PI3K and mTOR pathway with a potent anti-proliferative activity.Objective:We set out to evaluate the efficacy of GDC-0980, in pre-clinical studies, against pediatric leukemia cells.Methods:The anti-neoplastic activity of GDC-0980 was evaluated in vitro using five different pediatric leukemia cells.Results:Our data show that GDC-0980 significantly inhibited the proliferation of leukemia cell lines, KOPN8 (IC50, 532 nM), SEM (IC50,720 nM), MOLM-13 (IC50,346 nM), MV4;11 (IC50,199 nM), and TIB-202 (IC50, 848 nM), compared to normal control cells (1.23 µM). This antiproliferative activity was associated with activation of cellular apoptotic mechanism characterized by a decrease in Bcl-2 protein phosphorylation and enhanced PARP cleavage. Western blot analyses of GDC-0980 treated cells also showed decreased phosphorylation levels of mTOR, Akt and S6, but not ERK1/2. Notably, FLT3 phosphorylation was decreased in Molm-13 and MV4;11 cells following the application of GDC-0980. We further examined cellular viability of GDC-0980-treated primary leukemia cells isolated from pediatric leukemia patients. This study revealed a potential therapeutic effect of GDC-0980 on two ALL patients (IC50’s, 1.23 and 0.625 µM, respectively). Drug combination analyses of GDC-0980 demonstrated a synergistic activity with the MEK inhibitor Cobimetinib (MV4-11; 11, CI, 0.25, SEM, CI, 0.32, and TIB-202, CI, 0.55) and the targeted FLT3 inhibitor, Crenolanib (MV4-11; 11, CI, 0.25, SEM, CI, 0.7, and TIB-202, CI, 0.42).Conclusion:These findings provide initial proof-of-concept data and rationale for further investigation of GDC-0980 in selected subgroups of pediatric leukemia patients.

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