scholarly journals AVA4746, an Orally Available, Clinical Grade Antagonist of Integrin Alpha 4, Sensitizes Pre-B Cell Acute Lymphoblastic Leukemia to Chemotherapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2765-2765 ◽  
Author(s):  
Yongsheng Ruan ◽  
Eun Ji Gang ◽  
Hye-Na Kim ◽  
Chintan Parekh ◽  
Hisham Abdel-Azim ◽  
...  
Keyword(s):  
Overall Survival ◽  
Drug Resistance ◽  
Acute Lymphoblastic Leukemia ◽  
Metabolic Activity ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Drug Resistant ◽  
Support Research

Abstract Background. Even though remarkable progress has been made in the treatment of childhood acute lymphoblastic leukemia (ALL), salvage of relapse patients remains a challenge. The role of the bone marrow (BM) microenvironment is critical to protect leukemia cells from chemotherapy. The BM microenvironment promotes cell adhesion-mediated drug resistance (CAM-DR) in ALL.We and others have shown that the adhesion molecule integrin α4, referred to hereafter as α4, mediates drug resistance of B-ALL. In our previous studies, we showed that both α4 blockade by natalizumab and inhibition by the small molecule α4 antagonist TBC3486 can sensitize relapsed ALL cells to chemotherapy. However, no α4 targeting therapy is currently clinically available to treat leukemia. Here, we preclinically evaluate a novel non-peptidic small molecule antagonist, AVA4746, which has been safely used in clinical studies, as a potential new approach to combat drug resistant ALL. Method. Six refractory or relapsed primary pre-B ALL cases were used for in vitro studies. Viability was assessed by trypan blue counts or annexin V/7AAD flow cytometric analysis and metabolic activity was evaluated by Cytoscan WST-1 assay. For in vivo evaluation a NOD/SCID IL2Rγ-/- xenograft model of primary pre-B ALL (LAX7R) was used.AVA4746 (15mg/kg) was administered by oral gavage twice a day continuously for 14 days, and vincristine, dexamethasone, L-asparaginase (VDL) was given intraperitoneally (weekly) for 4 weeks. Overall survival was determined by Kaplan-Meier Survival analysis. Results. AVA4746 caused a significant decrease in mean fluorescence intensity (MFI) of α4 expression in six out of six ALL cases at doses of both 5μM and 25μM after 24 hours and 96 hours compared to DMSO control. Interestingly, decreased protein expression of α4 was also observed by Western Blot analysis all six ALL cases. We tested next in two cases (LAX53, ICN13), if AVA4746 de-adheres ALL cells from its counter receptor VCAM-1. The percentages of adherence after treatment with AVA4746 (25μM) were significantly lower than after DMSO treatment (10.3%±4.9% vs. 99.9%±7.6%, p= 0.00007 for LAX7R; 8.1%±1.0% vs. 100.1%±13.6%, p= 0.0003 for LAX53; 9.0%±1.6% vs. 100.0%±14.0%, p=0.0004 for ICN13). AVA4746 was not associated with apoptosis in vitro alone or in combination with chemotherapy (VDL). Metabolic activity as assessed by WST-1 assay was markedly decreased by AVA4746 in two of two ALL cases. AVA4746 also decreased ALL proliferation in two out of two ALL samples tested. In vivo, AVA4746 in combination with VDL chemotherapy treatment led to significant prolongation of overall survival (n=6) compared with the VDL only treated group (n=6) (MST= 78.5 days vs MST= 68 days; P<0.05). There was no significant difference in survival between the PBS control group (n=5) and the AVA4746 mono-treatment group (n=5) (MST=38days vs MST= 38days). Conclusion. We have identified α4 as a central adhesion molecule in CAM-DR of ALL and have shown that AVA-4746, an orally available and specific α4 antagonist, which has been safely used in clinical studies, downregulates α4 in primary ALL and functionally de-adheres them from VCAM-1. Critically, we demonstrated that inhibition of α4 in combination with standard chemotherapy can prolong the survival of NSG mice bearing pre-B ALL. These data support further study of inhibition of α4 using AVA4746 as a novel strategy to treat drug resistant B lineage ALL. Disclosures Bhojwani: Amgen: Other: Blinatumumab global pediatric advisory board 2015. Wayne:Spectrum Pharmaceuticals: Honoraria, Other: Travel Support, Research Funding; Kite Pharma: Honoraria, Other: Travel support, Research Funding; Pfizer: Consultancy, Honoraria, Other: Travel Support; Medimmune: Honoraria, Other: Travel Support, Research Funding; NIH: Patents & Royalties. Kim:Antisense Therapeutics Ltd: Patents & Royalties.

scholarly journals Tirapazamine As a Strategy to Overcome Hypoxia-Induced Drug Resistance in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4436-4436
Author(s):  
Barbara Muz ◽  
Pilar De La Puente ◽  
Micah John Luderer ◽  
Farideh Ordikhani ◽  
Abdel Kareem Azab
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Drug Resistance ◽  
Cell Survival ◽  
Research Funding ◽  
High Dose ◽  
Drug Resistant ◽  
Hypoxic Conditions

Abstract Introduction: Multiple myeloma (MM) is a lymphoplasmacytic malignancy characterized by the continuous spread of MM cells in and out of the bone marrow (BM). Despite the introduction of novel therapies, cancer patients relapse due to the development of drug resistant cells, which are, at least in part, promoted by hypoxia. Therefore, in this study we aimed to overcome drug resistance in MM by inhibition of the hypoxic responses in these cells. Tirapazamine (TPZ) is a hypoxia-activated pro-drug causing cell apoptosis, which has been shown to improve the outcome of patients with solid tumors when combined with radiotherapy; however, it has not been tested in MM. We used TPZ for the first time in MM to target the drug resistant cancer cells and sensitize them to therapy. Methods: To test the effect of TPZ on tumor survival in vitro, MM cell lines (MM1.s, H929, OPM1, RPMI8226) were exposed to normoxia (21% O2) or hypoxia (1% O2) for 24 hours with different concentrations of TPZ in order to obtain an IC50, and cell survival was assessed using MTT assay. Also, a combination of bortezomib and carfilzomib with or without TPZ was tested on cell survival. For in vivo study, 5 x 106 MM1s-Luc-GFP cells were injected intravenously (IV) into SCID mice and tumor progression was monitored for 3 weeks by bioluminescent imaging. First, we tested the hypoxic status of mice treated with and without a high-dose bortezomib (1.5mg/kg). Pimonidazole (PIM) was injected intraperitoneally (IP) into mice and 4 hours later BM was harvested, stained with anti-PIM-APC antibody and followed by measuring PIM signal in MM1s-GFP+ cells using flow cytometry. Second, we developed drug resistant cells by treating mice with a high-dose bortezomib (1.5mg/kg), and then treated with (1) bortezomib only (0.5mg/kg; n=3), or (2) bortezomib and TPZ (40mg/kg; n=3), all administered IP sequentially twice a week. The number of residual MM1s-GFP+ cells was calculated by flow cytometry. Results: We found that TPZ was active in a dose-dependent manner only in hypoxic conditions in MM cell lines. We showed that residual MM cells in the BM after high-dose bortezomib are hypoxic, as demonstrated by PIM staining. The combination of TPZ with bortezomib and carfilzomib resensitized cancer cells to death in hypoxia, overcoming hypoxia-induced drug resistance in vitro. Moreover, TPZ-treatment in combination with bortezomib further decreased residual MM cells in vivo. Conclusions: We reported that MRD was hypoxic and that TPZ, which was cytotoxic for MM cells only in hypoxic conditions, overcame hypoxia-induced drug resistance in vitro and killed bortezomib-resistant residual MM cells in vivo. This is the first study to show the efficacy of TPZ in MM. This data provides a preclinical basis for future clinical trials testing efficacy of TPZ in MM. Disclosures Azab: Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner ; Verastem: Research Funding.

scholarly journals Synthetic De Novo Modeling of Human T-Cell Acute Lymphoblastic Leukemia Reveals HOXB Genes Drive Expansion of Leukemia Cells-of-Origin and Established Tumor Clones

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1322-1322
Author(s):  
Manabu Kusakabe ◽  
Ann Chong Sun ◽  
Kateryna Tyshchenko ◽  
Rachel Wong ◽  
Aastha Nanda ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Research Funding ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Rna Seq ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Mechanistic studies in human cancer have relied heavily on established cell lines and genetically engineered mouse models, but these are limited by in vitro adaptation and species context issues, respectively. More recent efforts have utilized patient-derived xenografts (PDX); however, as an experimental model these are hampered by their variable genetic background, logistic challenges in establishing and distributing diverse collections, and the fact they cannot be independently reproduced. We report here a completely synthetic, efficient, and highly reproducible means for generating T-cell acute lymphoblastic leukemia (T-ALL) de novo by lentiviral transduction of normal CD34+ human cord blood (CB) derived hematopoietic progenitors with a combination of known T-ALL oncogenes. Transduced CB cells exhibit differentiation arrest and multi-log expansion when cultured in vitro on OP9-DL1 feeders, and generate serially transplantable, aggressive leukemia when injected into immunodeficient NSG mice with latencies as short as 80 days (median 161 days, range 79-321 days). RNA-seq analysis of synthetic CB leukemias confirmed their reproducibility and similarity to PDX tumors, while whole exome sequencing revealed ongoing clonal evolution in vivo with acquisition of secondary mutations that are seen recurrently in natural human disease. The in vitro component of this synthetic system affords direct access to "pre-leukemia" cells undergoing the very first molecular changes as they are redirected from normal to malignant developmental trajectories. Accordingly, we performed RNA-seq and modified histone ChIP-seq on nascently transduced CB cells harvested from the first 2-3 weeks in culture. We identified coordinate upregulation of multiple anterior HOXB genes (HOXB2-B5) with contiguous H3K27 demethylation/acetylation as a striking feature in these early pre-leukemia cells. Interestingly, we also found coordinate upregulation of these same HOXB genes in a cohort of 264 patient T-ALLs (COG TARGET study) and that they defined a subset of patients with significantly poorer event-free survival (Log-rank p-value = 0.0132). Patients in the "HOXB high" subgroup are distinct from those with ETP-ALL, but are enriched within TAL1, NKX2-1, and "unknown" transcription factor genetic subgroups. We further show by shRNA-mediated knockdown that HOXB gene expression confers growth advantage in nascently transduced CB cells, established synthetic CB leukemias, and a subset of established human T-ALL cell lines. Of note, while there is prior literature on the role of HOXA genes in AML and T-ALL, and of HOXB genes in normal HSC expansion, this is the first report to our knowledge of a role for HOXB genes in human T-ALL despite over 2 decades of studies relying mostly on mouse leukemia and cell line models. The synthetic approach we have taken here allows investigation of both early and late events in human leukemogenesis and delivers an efficient and reproducible experimental platform that can support functional testing of individual genetic variants necessary for precision medicine efforts and targeted drug screening/validation. Further, since all tumors including PDXs continue to evolve during serial propagation in vivo, synthetic tumors represent perhaps the only means by which we can explore early events in cellular transformation and segregate their biology from confounding effects of multiple and varied secondary events that accumulate in highly "evolved" samples. Disclosures Steidl: Seattle Genetics: Consultancy; Tioma: Research Funding; Bristol-Myers Squibb: Research Funding; Roche: Consultancy; Juno Therapeutics: Consultancy; Nanostring: Patents & Royalties: patent holding.

Functional Modulation of VLA6 in BCR-ABL1+ Pre-B Acute Lymphoblastic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2565-2565
Author(s):  
Eun Ji Gang ◽  
Yao-Te Hsieh ◽  
Huimin Geng ◽  
Jennifer Pham ◽  
Markus Muschen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Drug Resistance ◽  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Conditional Knockout ◽  
Leukemia Cells

Abstract Abstract 2565 Chemotherapy drug resistance in acute lymphoblastic leukemia (ALL) remains a major problem, resulting in reduced treatment efficacy and relapse. The bone marrow environment (BME) has been shown to promote resistance of leukemia cells towards chemotherapy, which has been attributed to several proteins, including integrins. Our analysis of 207 children with high-risk (BCR/ABL1−) pre-B ALL revealed that high expression of the laminin-binding integrin VLA6 (alpha6beta1) portends poor clinical outcomes in patients with minimal residual disease (MRD+) on day 29 of induction. In addition, our comparative analysis of pre-B leukemia and normal B-cells revealed that VLA6 is preferentially upregulated on BCR/ABL1+ pre-B ALL blasts. Alterations in adhesion properties have been described for BCR/ABL1+ (p210) chronic myeloid leukemia. The role of integrins and integrin VLA6 in particular for cell adhesion-mediated drug resistance (CAM-DR) in BCR/ABL1+ (p210) ALL has not been addressed. With respect to its role for normal immature hematopoietic cells, contradictory observations have been reported. Therefore, we hypothesized that VLA6-mediated adhesion of ALL cells to the bone marrow stromal niche contributes to drug resistance. We evaluated the role of VLA6 in BCR-ABL1+ leukemia using two of our established models of leukemia, a conditional knockout model of VLA6 in murine BCR-ABL1+ leukemia and a xenograft model of human BCR-ABL1+ leukemia. VLA6fl/fl cells were oncogenically transformed using BCR-ABL1 (p210) and cultured under lymphoid-skewing conditions. Induction of pre- B (B220+ CD19+) ALL was confirmed by flow cytometry. Subsequent transduction with CreERT2 or EmptyERT2 generated leukemia cells in which VLA6 ablation could be induced (CreERT2) or not (EmptyERT2) by addition of Tamoxifen. Conditional ablation of VLA6 in vitro decreased adhesion significantly compared to undeleted controls (19.7%±8.1% vs. 87.7%±6.0%; p=0.00041) and increased apoptosis of murine BCR-ABL1+ leukemia cells as determined by analysis of Annexin V−/7-AAD− viable cells by flow cytometry (VLA6 deleted vs. undeleted: 35.3%±1.1% vs. 75.1%±1.2%; p=0.0001). Moreover, VLA6 deletion sensitized murine ALL to a tyrosine kinase inhibitor (TKI), Nilotinib (p=0.022, 45.6%±2.4% vs. 73.3%±13.0%). To test the effect of VLA6 deletion on leukemic progression in vivo, VLA6 BCR/ABL1+ pre-B (B220+ CD19+) CreERT2+ or control transduced ALL cells were transferred into NOD/SCID mice. 3 days thereafter, VLA6 deletion was induced by Tamoxifen administration to all animals in 2 cycles for 5 days. In vivo deletion of VLA6 in delayed leukemia progression compared to VLA6 competent controls from a median survival time (MST) of 30 days post-leukemia injection to a MST of 43 days post-leukemia injection (p=0.008 Log-rank test). In vivo deletion of VLA6 in combination with Nilotinib treatment delayed leukemia progression compared to VLA6 competent, as Nilotinib-treated control animals have uniformly died of leukemia with a MST of 39.5 days, however the Nilotinib treated VLA6 deleted group is completely alive and is still being monitored (p=0.0025). When VLA6 was ablated before transfer and recipients were observed for leukemia progression, the recipients of VLA6–deficient murine leukemia cells also showed attenuated leukemia progression compared to recipients of VLA6 competent cells. Moreover, we show that VLA6 blockade de-adheres primary ALL cells from their cognate counter receptor laminin in vitro, and sensitizes primary ALL cells to TKI Taken together, modulating the function of VLA6 in ALL offers a new approach to overcome drug resistance in ALL. Given that VLA6 is probably largely redundant for normal immature hematopoiesis, this approach may be preferable over targeting of other integrins in BCR/ABL1+ leukemias on which VLA6 is expressed. Disclosures: No relevant conflicts of interest to declare.

Targeting Survivin In Recalcitrant Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3263-3263
Author(s):  
Eugene Park ◽  
Enzi Jiang ◽  
Yao-Te Hsieh ◽  
Lars Klemm ◽  
Cihangir Duy ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Treated Group ◽  
Locked Nucleic Acid ◽  
Drug Resistant ◽  
Loss Of Function ◽  
Self Renewal

Abstract Abstract 3263 Background: Despite the recent advances in chemotherapy for acute lymphoblastic leukemia (ALL), the development of drug resistance and long-term side effects of current treatments warrant new treatment modalities. Survivin/BIRC5, an inhibitor of apoptosis protein, is critical for the survival and proliferation of cancerous cells, is expressed in AML and ALL cells, and has been implicated in leukemia relapse. In the present study, we test the hypothesis that survivin is critical to the pathway of self-renewal of drug-resistant ALL cells. Methods: For gain of function studies, primary ALL cells were transduced with lentiviral survivin IRES GFP reporter (Survivin GFP) or empty GFP as a control. For loss of function studies, an inducible lentiviral shRNA vector expressing tRFP upon induction with doxycyclin was used. For in vitro evaluation of Survivin, CFU assays were used to monitor self-renewal capability, MTT assays and Trypan blue counts for viability determination were used for drug testing. For in vivo experiments, we used a NOD/SCID IL2Rγ−/- xenograft model with patient-derived ALL cells. Results: Survivin overexpression in primary ALL cells led in vitro to 4-fold more colonies than control in primary and secondary CFU assays and to increased resistance against Vincristine, Dexamethasone and L-Asparaginase (VDL) compared to controls (p<0.05). In vivo, animals injected with ALL cells overexpressing survivin died earlier of leukemia with a median survival time (MST) of 43 days (n=4) compared to control animals (MST=51.5 days) (n=4) (p<0.05). Therefore, overexpression of survivin increases self-renewal of patient-derived ALL cells in vitro and accelerates leukemia development in vivo. Conversely, in vitro inhibition of Survivin using shRNA decreased CFU compared to controls (p<0.0001). In vivo, when animals were injected with Survivin shRNA or non-silencing control shRNA and treated for 4 weeks with VDL, the combined VDL + Survivin shRNA treated group not only lived significantly longer (MST=213 days, n=3) compared to the control group (MST=117 days; n=3) (p<0.05) but remained disease-free until the end of follow-up (Day 213). Immunohistology and flow cytometry staining for huCD45+ cells in various organs showed the absence of leukemia cells in the VDL + shRNA treated group compared to the control, indicating that adjuvant knockdown of Survivin in combination with chemotherapy eradicates drug resistant primary leukemia. To further evaluate loss of function of Survivin in ALL, we used a survivin knockout mouse model. Survivinflox/flox bone marrow cells were retrovirally transformed with BCR-ABL1 p210 or MLL-ENL. Subsequent to leukemic outgrowth, cells were transduced with either GFP control or inducibly deleted using a Cre-GFP vector and GFP signal was quantitated by flow cytometry. Interestingly, Survivin deleted Cre-GFP positive oncogene transformed cells showed reduced proliferation compared to GFP controls (p<0.05), indicating that knockout of Survivin in murine leukemia is required for survival of leukemic cells. Finally, we determined the effect of pharmacological downregulation of Survivin using EZN-3042, a novel locked nucleic acid antisense oligonucleotide (LNA-AsODN) against Survivin. Single agent treatment of 6 primary ALL cases, with 20 mM of a scrambled LNA control (EZN-3088) or EZN-3042, were respectively assayed. Mean viability for EZN-3088 treatments was 78.6% ± 12.0% versus 44.1% ± 10.9% for EZN-3042 (p<0.001). EZN-3042 knockdown of Survivin was confirmed by Western blot. LNA treatment in combination with chemotherapy of a primary Philadelphia chromosome positive (Ph+) ALL resulted in a mean viability of 73.4% ± 1.8% for EZN-3088/Nilotinib versus 3.6% ± 0.5% for EZN-3042/Nilotinib (p<0.001). Primary Ph− ALL cells treated with EZN-3088/VDL resulted in a mean viability of 36.5% ± 0.5% versus 8.3% ± 4.3% for EZN-3042/Nilotinib (p<0.01). Conclusion: Taken together, we show that Survivin is a key component in primary drug resistant ALL cells and adjuvant specific targeting of Survivin using shRNA or EZN3042 has the potential to eradicate relapse of leukemia. Disclosures: Yang: Amgen Inc: Employment, Equity Ownership.

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

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

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

HSP90 Inhibition Targets JAK2 and Is Highly Effective in CRLF2-Rearranged Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 576-576
Author(s):  
Oliver Weigert ◽  
Liat Bird ◽  
Nadja Kopp ◽  
Andrew A. Lane ◽  
Bjoern Chapuy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Overall Survival ◽  
Acute Lymphoblastic Leukemia ◽  
Heat Shock ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Enrichment Analysis ◽  
Jak Inhibitor ◽  
Hsp90 Inhibition

Abstract Abstract 576 Enzymatic inhibitors of Janus kinase 2 (JAK2) are in clinical development for the treatment of B-cell acute lymphoblastic leukemia (B-ALL) with rearrangements of the cytokine receptor subunit CRLF2, myeloproliferative neoplasms, and other tumors with constitutive JAK2 signaling. In addition, JAK2 is a client of heat shock protein 90 (HSP90) and HSP90 inhibition results in JAK2 degradation. To explore the utility of blocking JAK2 in CRLF2-rearranged B-ALL, we exposed the MHH-CALL4 and MUTZ-5 cell lines, which both have CRLF2/IGH rearrangements and activating JAK2 mutations to a panel of JAK2 inhibitors (JAK inhibitor-1, INCB18424, tofacitinib, NVP-BSK805, NVP-BVB808, TG101348) and the HSP90 inhibitor NVP-AUY922. Both MUTZ-5 and MHH-CALL4 were highly sensitive to AUY922 (GI50, 25–26 nM), with 50- to >1,000-fold superior potency compared with the panel of JAK2 enzymatic inhibitors. AUY922 and the structurally divergent HSP90 inhibitors 17-AAG, PU-H71 and HSP990 were all potently active against a panel of Ba/F3 lines dependent on CRLF2 and JAK2 signaling (GI50, 1–11 nM). Treatment of MUTZ-5 and MHH-CALL4 cells with JAK inhibitor-1 reduced but did not eliminate phospho (P-) STAT5 and P-ERK1/2 in both lines but promoted increases in P-AKT in MUTZ-5 and P-JAK2 in MHH-CALL4. In contrast, AUY922 treatment more extensively reduced or eliminated phosphorylation of all the targets. The combination of AUY922+JAK inhibitor-1 had little or no additional effect on target phosphorylation compared with AUY922 alone and pairwise dose-response studies with isobologram analysis failed to identify synergistic effects. We performed transcriptional profiling on MUTZ-5 and MHH-CALL cells treated with either vehicle (DMSO), JAK inhibitor-1, AUY922 or JAK inhibitor-1+AUY922. Unsupervised hierarchical clustering distinguished samples treated with AUY922 (or combination) from those treated with JAK inhibitor-1 or vehicle. To formally assess whether AUY922 targets the same genes as JAK inhibitor-1, we defined a ‘JAK inhibitor signature' from the top/bottom 250 most differentially expressed genes following treatment with JAK inhibitor-1. Using gene set enrichment analysis (GSEA), the ‘JAK inhibitor signature' was highly enriched upon treatment with AUY922 (p=0.003). GSEA also demonstrated that STAT5A signatures were enriched upon treatment with JAK inhibitor-1, AUY922, or JAK inhibitor-1+AUY922. To identify additional targets of HSP90 inhibition beyond the inhibition of JAK2, we used the C3 database from the MsigDB compendium to perform a transcription factor binding site enrichment analysis on the most differentially expressed genes between JAK inhibitor-1 and AUY922. The top 5 hits were all heat-shock factors (HSF, FDR<0.05). GSEA revealed that an HSF1 signature was only enriched upon treatment with AUY922 or AUY922+JAKinh-1, but not after JAKinh-1 alone. To extend our findings to in vivo treatment of human B-ALL, we established primary B-ALL xenografts from CRLF2-rearranged, patient-derived bone marrow samples in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. To stringently assay established disease in vivo, we waited until bone marrow leukemia burden exceeded 30% and then initiated treatment with BVB808 50mg/kg twice daily, AUY922 50mg/kg thrice weekly, BVB808+AUY922 or vehicle. After 5 days of treatment, spleens from mice treated with vehicle or BVB808 had nearly complete effacement by B-ALL, while AUY922 or BVB808+AUY922 treatment resulted in visible islands of hematopoiesis. Based on immunohistochemistry, mice receiving AUY922 or BVB808+AUY922, but not BVB808 or vehicle, had nearly complete loss of P-STAT5 as well as upregulation of the pharmacodynamic marker HSP70. Immunoblotting of spleens from treated mice demonstrated reductions in P-STAT5, P-JAK2, and total JAK2 in AUY922- or BVB808+AUY922-treated mice. In contrast, treatment with single-agent BVB808 only modestly suppressed P-STAT5. Treatment with either BVB808 or AUY922 prolonged overall survival compared to vehicle (p=0.01 for both xenografts). Treatment with AUY922 further prolonged overall survival compared to BVB808 (p<0.01 for both xenografts), while the combination of BVB808 and AUY922 had no additional benefit compared to AUY922 alone. In conclusion, HSP90 is a promising therapeutic target in CRLF2-rearranged B-ALL and merits clinical evaluation. Disclosures: Romanet: Novartis Pharma AG: Employment. Murakami:Novartis Pharma AG: Employment. Sallan:Enzon Pharmaceuticals: Honoraria. Kung:Novartis Pharmaceuticals: Consultancy, Research Funding. Radimerski:Novartis Pharma AG: Employment. Weinstock:Novartis: Consultancy, Research Funding.

scholarly journals BRD4 PROTAC degrader ARV-825 inhibits T-cell acute lymphoblastic leukemia by targeting 'Undruggable' Myc-pathway genes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shuiyan Wu ◽  
You Jiang ◽  
Yi Hong ◽  
Xinran Chu ◽  
Zimu Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Rna Seq ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bet Protein

Abstract Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a high risk of induction failure and poor outcomes, with relapse due to drug resistance. Recent studies show that bromodomains and extra-terminal (BET) protein inhibitors are promising anti-cancer agents. ARV-825, comprising a BET inhibitor conjugated with cereblon ligand, was recently developed to attenuate the growth of multiple tumors in vitro and in vivo. However, the functional and molecular mechanisms of ARV-825 in T-ALL remain unclear. This study aimed to investigate the therapeutic efficacy and potential mechanism of ARV-825 in T-ALL. Methods Expression of the BRD4 were determined in pediatric T-ALL samples and differential gene expression after ARV-825 treatment was explored by RNA-seq and quantitative reverse transcription-polymerase chain reaction. T-ALL cell viability was measured by CCK8 assay after ARV-825 administration. Cell cycle was analyzed by propidium iodide (PI) staining and apoptosis was assessed by Annexin V/PI staining. BRD4, BRD3 and BRD2 proteins were detected by western blot in cells treated with ARV-825. The effect of ARV-825 on T-ALL cells was analyzed in vivo. The functional and molecular pathways involved in ARV-825 treatment of T-ALL were verified by western blot and chromatin immunoprecipitation (ChIP). Results BRD4 expression was higher in pediatric T-ALL samples compared with T-cells from healthy donors. High BRD4 expression indicated a poor outcome. ARV-825 suppressed cell proliferation in vitro by arresting the cell cycle and inducing apoptosis, with elevated poly-ADP ribose polymerase and cleaved caspase 3. BRD4, BRD3, and BRD2 were degraded in line with reduced cereblon expression in T-ALL cells. ARV-825 had a lower IC50 in T-ALL cells compared with JQ1, dBET1 and OTX015. ARV-825 perturbed the H3K27Ac-Myc pathway and reduced c-Myc protein levels in T-ALL cells according to RNA-seq and ChIP. In the T-ALL xenograft model, ARV-825 significantly reduced tumor growth and led to the dysregulation of Ki67 and cleaved caspase 3. Moreover, ARV-825 inhibited cell proliferation by depleting BET and c-Myc proteins in vitro and in vivo. Conclusions BRD4 indicates a poor prognosis in T-ALL. The BRD4 degrader ARV-825 can effectively suppress the proliferation and promote apoptosis of T-ALL cells via BET protein depletion and c-Myc inhibition, thus providing a new strategy for the treatment of T-ALL.

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

Blood ◽  
2020 ◽  
Vol 136 (2) ◽  
pp. 210-223 ◽  
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.

scholarly journals Phase I Study of Ixazomib Added to Chemotherapy in the Treatment of Acute Lymphoblastic Leukemia in Older Adults

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 41-42
Author(s):  
Philip C. Amrein ◽  
Karen K. Ballen ◽  
Kristen E. Stevenson ◽  
Traci M. Blonquist ◽  
Andrew M. Brunner ◽  
...  
Keyword(s):  
Overall Survival ◽  
Acute Lymphoblastic Leukemia ◽  
Complete Remission ◽  
Board Of Directors ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Advisory Committees ◽  
Survival Estimate ◽  
Grade 3 ◽  
Experienced Grade

Introduction: While progress has been made in the treatment of childhood leukemia, the outlook for patients &gt;60 years of age with acute lymphoblastic leukemia (ALL) is poor with complete remission rates (CR) of approximately 60% and 3-year survivals (OS) of less than 15%. Intensified treatment in a later CALGB trial showed little improvement with a CR=61% and 5-year OS=6% (Stock, Cancer 2013). Ixazomib is an oral proteasome inhibitor, which has shown single agent activity and promising combination activity in pediatric ALL patients (Messinger, Blood 2012). We sought to assess the safety and tolerability, as well as early efficacy of adding ixazomib to a current MGH-DFCI/HCC multi-agent regimen for older adults with ALL. Methods: Patients aged 51 to 75 years of age with newly diagnosed B-ALL and T-ALL were screened for eligibility. Patients with mature ALL (including Burkitt's) were excluded. Patients with Philadelphia chromosome positive ALL (BCR-ABL1+) were eligible, and dasatinib was added to the chemotherapy on Day 10 for these patients. The chemotherapy treatment schedule from induction through maintenance is outlined in Table 1. A standard 3 + 3 patient cohort dose escalation design was used to determine the maximum tolerated dose (MTD) of ixazomib during induction for these patients, the primary objective of the trial. After consolidation I, patients in complete remission (CR) with a suitable donor were offered a hematopoietic stem cell transplantation (HSCT) as per institutional guidelines. Those not going to HSCT continued therapy as noted in the table. Results: There were 19 patients with B-ALL enrolled, none with T-ALL. Among these patients, 7 harbored BCR-ABL1 rearrangements. The median age was 65 years, 74% were male, and 90% had a performance status 0 or 1. The MTD was 2.3 mg of ixazomib, as 2 patients at 3.0 mg developed DLT's: a grade 3 peripheral neuropathy and a grade 5 acute kidney injury (Table 2). Grade 3 and 4 toxicities encountered at any time consisted mainly of grade 4 neutropenia in 13 patients and grade 4 thrombocytopenia in 12 patients. One patient experienced grade 3 neutropenia and 5 patients experienced grade 3 thrombocytopenia. Two patients with grade 2 neuropathy did not meet the definition of DLT. Among the 19 patients, 15 (79%, [95% confidence interval (CI), 54-94%]) achieved CR (14) or CRi (1), and 5 patients went on to HSCT. The median follow-up time was 2 years (range, 1-5) for 8 patients remaining alive. The 1-year overall survival estimate was 53% [95% CI, 29-72%], while the 2-year overall survival estimate was 47% [95% CI, 24-67%]. Conclusions: A dose of 2.3 mg of ixazomib in combination with induction chemotherapy among older patients with ALL was well-tolerated and associated with a promising rate of complete remission. Disclosures Amrein: Takeda: Research Funding; AstraZeneca: Consultancy, Research Funding; Amgen: Research Funding. Brunner:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Research Funding; AstraZeneca: Research Funding; Forty-Seven Inc: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding. Hobbs:Novartis: Honoraria; Celgene/BMS: Honoraria; Jazz: Honoraria; Constellation: Honoraria, Research Funding; Incyte: Research Funding; Merck: Research Funding; Bayer: Research Funding. Neuberg:Celgene: Research Funding; Pharmacyclics: Research Funding; Madrigak Pharmaceuticals: Current equity holder in publicly-traded company. Fathi:Takeda: Consultancy, Research Funding; Agios: Consultancy, Research Funding; PTC Therapeutics: Consultancy; Amphivena: Consultancy; Astellas: Consultancy; Daiichi Sankyo: Consultancy; Novartis: Consultancy; Newlink Genetics: Consultancy; Pfizer: Consultancy; Blueprint: Consultancy; Trillium: Consultancy; Kura Oncology: Consultancy; Forty Seven: Consultancy; Jazz: Consultancy; Boston Biomedical: Consultancy; BMS/Celgene: Consultancy, Research Funding; Kite: Consultancy; Trovagene: Consultancy; Amgen: Consultancy; Seattle Genetics: Consultancy, Research Funding; Abbvie: Consultancy. OffLabel Disclosure: MLN 9708, ixazomib is FDA approved for multiple myeloma. In this trial it is used to treat acute lymphoblastic leukemia.

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