BAP1 regulates epigenetic switch from pluripotency to differentiation in developmental lineages giving rise to BAP1-mutant cancers

The BAP1 tumor suppressor is mutated in many human cancers such as uveal melanoma, leading to poor patient outcome. It remains unclear how BAP1 functions in normal biology or how its loss promotes cancer progression. Here, we show that Bap1 is critical for commitment to ectoderm, mesoderm, and neural crest lineages during Xenopus laevis development. Bap1 loss causes transcriptional silencing and failure of H3K27ac to accumulate at promoters of key genes regulating pluripotency-to-commitment transition, similar to findings in uveal melanoma. The Bap1-deficient phenotype can be rescued with human BAP1, by pharmacologic inhibition of histone deacetylase (HDAC) activity or by specific knockdown of Hdac4. Similarly, BAP1-deficient uveal melanoma cells are preferentially vulnerable to HDAC4 depletion. These findings show that Bap1 regulates lineage commitment through H3K27ac-mediated transcriptional activation, at least in part, by modulation of Hdac4, and they provide insights into how BAP1 loss promotes cancer progression.Fil: Kuznetsov, Jeffim N.. University of Miami; Estados UnidosFil: Agüero, Tristán Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. University of Miami; Estados UnidosFil: Owens, Dawn A.. University of Miami; Estados UnidosFil: Kurtenbach, Stefan. University of Miami; Estados UnidosFil: Field, Matthew G.. University of Miami; Estados UnidosFil: Durante, Michael A.. University of Miami; Estados UnidosFil: Rodriguez, Daniel A.. University of Miami; Estados UnidosFil: King, Mary Lou. University of Miami; Estados UnidosFil: Harbour, J. William. University of Miami; Estados Unido

AMPK and Akt Differentially Regulate the UPR in Acute Lymphoblastic Leukemia: Therapeutic Implications.

Acute Lymphoblastic Leukemia (ALL) is the most common hematological malignancy and the main cause of cancer-related death in children. Current chemotherapy treatments based mainly on antifolate regimens have lead to high cure rates (≈80%), however event-free survival (EFS) for children and adults diagnosed with chemotherapy resistant phenotypes of ALL or after the relapse, continues to be dismal (EFS ∼10-20%). High-risk chemotherapy treatment based on intensification strategies and/or the use of stem cell transplantation have led to marginal improvements with limited impact on cure rates of resistant/refractory or relapsed ALL phenotypes. Our studies attempted to discover highly effective treatment strategies for ALL by targeting novel molecular pathways with existing agents that represent a backbone of current effective chemotherapeutic regimens for ALL. Our findings for the first time demonstrated that ALL exhibited sensitivity to reactive oxygen species (ROS) -induced endoplasmic reticulum (ER) stress and the unfolded protein response (UPR)-mediated cell death. These findings suggest a novel use for methotrexate (MTX), the backbone of current antifolate-based ALL chemotherapy, in combination with exogenous 5-aminoimidazole- 4-carboxamide-1-beta-4-ribofuranoside (AICAr) as part of the treatment intensification to target ER stress/UPR signaling in ALL. The sensitivity of ALL cell lines and primary patient cells to ER stress-induced cytotoxicity was further confirmed by using the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) in ALL cell models. We found that 2-DG induced significant cell death under normoxia in Bp-ALL and T-ALL cells. This finding makes ALL one of the few tumor cell lines that exhibit sensitivity to glycolytic inhibitor 2-DG under normoxia. Using D-mannose, a sugar essential for N-linked glycosylation, our laboratory has postulated that 2-DG inhibits N-linked glycosylation and interferes with glycan-dependent folding and quality control of nascent ER proteins, which leads to ER stress-induced cytotoxicity in ALL. Our data also demonstrate for the first time that Akt and AMP-activated protein kinase (AMPK) differentially modulate the UPR function in ALL and determine the cellular responses (survival vs. death) depending on a type of ER stress-causing chemotherapeutic injury. In the case of AICAr plus MTX treatment we postulate that AMPK blocks the induction of the proapoptotic UPR signaling caused by the accumulation of ROS and the prolonged ER stress. In that case, Akt, a cellular antagonist of AMPK, plays a paradoxical proapoptotic role. However, in the case of 2- DG treatment, the effects of AMPK and Akt on cell cytotoxicity are reversed. We postulate that it is AMPK-mediated inhibition of the pro-survival glucose regulated protein 78 (GRP78)-regulated UPR signaling that synergistically potentiates apoptosis in ALL via induction of proteotoxicity. Our data underscore the complexity of the interactions within these pathways, and suggests that the contextual relationship between these signaling proteins and cross-talk with related pathways are critical determinants of cellular responses and fate in ALL cells

Abstract 5089: AMPK activationvs.inhibition induces apoptosis in acute lymphoblastic leukemia cells by differentially altering PI3K/Akt/mTOR or RAS/cRAF/MEK/Erk signaling

Abstract Acute Lymphoblastic Leukemia (ALL) is the most common hematological malignancy and the main cause of cancer-related deaths in children. Therefore, search for novel treatment strategies is warranted. We identified AMP activated protein kinase (AMPK), a master regulator of bioenergetics, as a potential target for ALL therapy due to its effects on cell proliferation and cell cycle regulation, as well as its crosstalk with critical metabolic and oncogenic pathways. We demonstrated that treatment of NALM6 (Bp-ALL) and CEM (T-ALL) cells with AICAR, an AMPK activator, induced growth inhibition and apoptosis. Using metformin, another AMPK agonist, we found 40% growth inhibition, and up to five- and three-fold greater induction of apoptosis relative to controls in CEM and NALM6, respectively. Unexpectedly, rescue experiments with AMPK inhibitors Ara-A and compound-C (CC) failed to abrogate the cytotoxic effects induced by AICAR. When used alone, Ara-A induced 60% and 40% cell death in NALM6 and CEM cells, respectively, whereas CC induced 75- and 15-fold more apoptotic death relative to controls. To investigate the mechanism by which AMPK activation vs. inhibition induced apoptosis, we determined levels of P-AMPK (T172) and factors associated with the PI3K/Akt/mTOR and RAS/cRAF/Erk signaling pathways in NALM6 and CEM cells treated with either CC, AICAR, or in combination. Our data show that P-AMPK levels were decreased by CC and increased by AICAR. Additional Western blots demonstrated that these agents exerted opposite effects on Akt and RAS signaling. CC decreased P-Akt (S473) and activated the RAS pathway, while AICAR increased P-Akt. We showed that activation of Akt by AICAR down-regulated the RAS pathway via phosphorylation of cRAF (S259). P-mTOR (S2448) and P-4EBP1 (T70) exhibited a greater decrease in cells treated with CC + AICAR as compared to each agent alone. A significant decrease in P-Akt was also detected in cells treated with both agents vs. each drug alone. Together, our data indicate that AICAR and CC induce cell death in ALL cells by two different mechanisms mediated by AMPK: AICAR-activation of AMPK inhibited the RAS-dependent cell proliferation pathway, and CC-inhibition of AMPK by down-regulating the Akt cell survival pathway. These results suggest that alterations in AMPK signaling may regulate the cross-talk between the PI3K/Akt/mTOR and RAS/cRAF/Erk cascades and may dictate the fate of ALL cells by regulating apoptosis after exposure to agents targeting these pathways. Experiments co-targeting AMPK and Akt using AICAR and Akt-inhibitor X, respectively, induced synergistic growth inhibition in CEM (CI=0.90) and NALM6 (CI=0.85) cells compared to each drug alone. These findings provide a rationale for simultaneously targeting AMPK and key signaling factors associated with either PI3K/Akt/mTOR or RAS/cRAF/Erk pathways in ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5089.</jats:p

Abstract 2736: PIM2 is up-regulated in response to metformin-induced cell death triggered by ER stress/UPR in ALL lymphoblasts

Abstract Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, and despite significant overall improvements in cure rates, outcome for patients diagnosed with resistant phenotypes or those who relapse is dismal. We investigated the mechanism of cell death induced by metformin (MET) in ALL cell models. We showed that MET induced significant growth inhibition and apoptosis in CCRF-CEM (T-ALL) and NALM6 (Bp-ALL) cells. Western blots revealed that MET activated p-AMPK, p-ACC, p-Akt, and p-4EBP1. The latter suggested that regulation of protein translation may be an important determinant in MET-induced cell death. Indeed, inhibition of mTOR/protein translation with rapamycin (RAPA) rescued MET-induced cell death in ALL cells. In addition, knockdown of AMPK expression in ALL cells using shRNA (shAMPK) abrogated MET-induced apoptosis as compared to control cells expressing scramble shRNA (shCTRL), indicating that AMPK mediated MET's cytotoxicity. Western blots demonstrated that shAMPK cells expressed lower level of total AMPK, p-p38MAPK, p-mTOR, and p-4EBP1 compared to shCTRL cells, indicating that AMPK and protein translation are critical in MET sensitivity. Indeed, pulse labeled 35S-methionine experiments demonstrated increase incorporation confirming the importance of protein synthesis is MET-induced cytotoxicity. More important, we uncovered that MET-induced apoptosis correlated with induction of ER stress as evidenced by up-regulation of IRE1α and CHOP although GRP78 decreased significantly. We further demonstrated that RAPA rescued MET-treated cells by relieving ER stress/UPR mediated cell death. We previously showed that the unfolded protein response (UPR) in ALL cells is regulated by the contextual crosstalk between AMPK and Akt (Mol Cancer Ther 10:437, 2011). Therefore, we evaluated the effects of co-targeting Akt and AMPK using the Akt inhibitor X/perifosine + MET and found these combinations to be synergistic. To further investigate the relationship between protein translation and ER stress/UPR in MET-induced cell death, we examined the role of PIM1/2 kinases. We found that expression of PIM2 was increased in MET-treated ALL with concomitant decreased in the expression of IRE1α, ATF6, and CHOP, suggesting that PIM2 maybe up-regulated as a compensatory survival mechanism aimed at relieving MET-induced ER stress/UPR mediated cell death. To test this hypothesis, we co-treated ALL cells with a PIM1/2 kinase inhibitor + MET and found that PIM2 inhibition synergistically sensitized ALL cells to MET (CI=0.28). Taken together, our data indicate that PIM2 plays a role in buffering cell death in MET-treated cells, and that regulation of protein translation modulates ER stress/UPR induced apoptosis in ALL cells. Consequently, our data support strategies targeting these synthetic lethal interactions as suitable for clinical translation in patients with ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2736. doi:1538-7445.AM2012-2736</jats:p

AMPK and Akt determine apoptotic cell death following perturbations of one-carbon metabolism by regulating ER stress in Acute Lymphoblastic Leukemia

AICAr is a cell-permeable nucleotide that has been used in vivo and in vitro to activate AMPK. Our previous findings have shown that AICAr as a single agent induces dose- and time-dependent growth inhibition in acute lymphoblastic leukemia (ALL) cell lines. In addition, the combination of AICAr with antifolates (methotrexate (MTX) or pemetrexed) has been shown to further potentiate AMPK activation and to lead to greater cytotoxicity and growth inhibition in leukemia and other malignant cell types. Our data presented herein demonstrate that sustained ER stress is the predominant mechanism behind the synergistic induction of cell death by the combination of AICAr plus the inhibitor of one-carbon metabolism, MTX, in Bp- and T-ALL, as evidenced by induction of several unfolded protein response markers leading to apoptosis. We also show for the first time that AICAr in combination with MTX significantly induces Akt phosphorylation in ALL. Under these conditions, the concomitant inhibition of Akt, a cellular antagonist of AMPK, leads to further up-regulation of AMPK activity and alleviates AICAr plus MTX-induced ER stress and apoptosis. Therefore, we also demonstrate that the concomitant activation of AMPK actually rescues the cells from AICAr plus MTX-induced ER stress and apoptosis. Our data suggest that the effects of AMPK activation on cell death or survival differ contextually depending on its signaling alterations with related oncogenic pathways and provide insight into the reported paradoxical pro-apoptotic vs. pro-survival effects of AMPK activation

PIM2 Upregulation Leads to Physiological Buffering of Metformin-Induced Cell Death Mediated by ER Stress/UPR in Acute Lymphoblastic Leukemia

Abstract Abstract 3496 Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in children and adolescents. Despite significant overall improvements in cure rates, outcome remains dismal for patients with resistant phenotypes or after relapse. Therefore, novel treatment strategies are warranted. Recently, we identified the AMP activated protein kinase (AMPK), a regulator of energy homeostasis in eukaryotic cells, as a potential target for ALL therapy due to its effects on cell growth, proliferation, and cell cycle regulation, as well as its crosstalk with critical metabolic and oncogenic pathways. We showed that activation of AMPK using metformin (1-5 mM) induced significant cell growth inhibition and apoptosis in CCRF-CEM (T-ALL) and NALM6 (Bp-ALL) cell line models. Western blot analysis revealed that metformin led to activation of p-AMPK (Thr172) and its downstream target p-ACC (Ser79), the cell proliferation regulator p-Akt (Ser473), and the protein translation regulator p-4EBP1 (Thr70), suggesting that protein translation may be an important determinant in the mechanism of metformin-induced cell death. Indeed, we demonstrated that blocking protein translation with the mTOR inhibitor rapamycin (1 μg/ml) rescued ALL cells from metformin-induced cell death (p < 0.01). In addition, knockdown of AMPK1α expression using shRNAs (shAMPK) abrogated metformin-induced growth inhibition and apoptosis in ALL cells as compared to control cells expressing scramble shRNAs (shCTRL), indicating that AMPK mediates metformin's cytotoxicity in our models. Western blots demonstrated that ALL cells expressing shAMPK exhibit decreased expression of total AMPK, p-p38MAPK (Thr180), p-mTOR (Ser2448), and p-4EBP1 (Thr70) compared to shCTRL cells, implicating regulation of protein translation in the mechanism of cell death induced by metformin. In addition, metformin-induced p-Akt (Ser473) activation observed in shCTRL cells is blocked in shAMPK expressing cells, suggesting that the contextual crosstalk between AMPK and Akt is relevant for metformin's cytotoxicity. Indeed, experiments co-targeting Akt and AMPK using perifosine (6 μM) or the Akt inhibitor X (AIX, 5 μM) plus metformin (5 mM) for 72 h induced synergistic cell death in NALM6 cells (Combination Index (CI) values of 0.21 for perifosine + metformin, and 0.19 for AIX + metformin). Our studies uncovered that apoptotic death in NALM6 and CCRF-CEM cells treated with metformin correlated with metformin's induction of ER stress/UPR in ALL cells, as demonstrated by increased expression of the UPR markers IRE1α and CHOP. More important, rapamycin rescued metformin-treated ALL cells by relieving ER stress/UPR as demonstrated by decreased IRE1α and CHOP. These observations support our previous findings that ER stress/UPR mediates cell death in ALL cells under metabolic stress, and is tightly coupled to regulation of protein translation (Mol Cancer Ther 10:437, 2011). To further investigate the relationship between protein translation and ER stress/UPR, we examined the role of PIM1/2 kinases, particularly PIM2 known to regulate CAP protein translation, in metformin-induced ALL cell death. Our results indicate that expression of PIM2 is significantly increased in NALM6 cells treated with metformin (5–10 mM) for 72 h. We also observed concomitant decrease in the expression of the UPR markers IRE1α, ATF6, and CHOP, raising the possibility that PIM2 upregulation may be a compensatory survival mechanism to regulate protein translation and suppress metformin-induced ER stress/UPR. To test this hypothesis, we co-treated NALM6 cells with the small molecule PIM1/2 kinase inhibitor V (80 μM) and metformin (5 mM) and found that inhibition of PIM2 in metformin-treated NALM6 cells induced synergistic cell death (CI = 0.28). Taken together, our data indicate that PIM2 plays a role in buffering cell death in metformin treated cells, and that regulation of protein translation modulates ER stress/UPR induced apoptosis in ALL cells. Consequently, our data support strategies that exploit synthetic lethality by combining activators of AMPK such as metformin and compounds that target regulation of protein translation or protein degradation as suitable for clinical translation in patients with ALL. Disclosures: No relevant conflicts of interest to declare

Metformin induces apoptosis through AMPK-dependent inhibition of UPR signaling in ALL lymphoblasts.

The outcome of patients with resistant phenotypes of acute lymphoblastic leukemia (ALL) or those who relapse remains poor. We investigated the mechanism of cell death induced by metformin in Bp- and T-ALL cell models and primary cells, and show that metformin effectively induces apoptosis in ALL cells. Metformin activated AMPK, down-regulated the unfolded protein response (UPR) demonstrated by significant decrease in the main UPR regulator GRP78, and led to UPR-mediated cell death via up-regulation of the ER stress/UPR cell death mediators IRE1α and CHOP. Using shRNA, we demonstrate that metformin-induced apoptosis is AMPK-dependent since AMPK knock-down rescued ALL cells, which correlated with down-regulation of IRE1α and CHOP and restoration of the UPR/GRP78 function. Additionally rapamycin, a known inhibitor of mTOR-dependent protein synthesis, rescued cells from metformin-induced apoptosis and down-regulated CHOP expression. Finally, metformin induced PIM-2 kinase activity and co-treatment of ALL cells with a PIM-1/2 kinase inhibitor plus metformin synergistically increased cell death, suggesting a buffering role for PIM-2 in metformin's cytotoxicity. Similar synergism was seen with agents targeting Akt in combination with metformin, supporting our original postulate that AMPK and Akt exert opposite regulatory roles on UPR activity in ALL. Taken together, our data indicate that metformin induces ALL cell death by triggering ER and proteotoxic stress and simultaneously down-regulating the physiologic UPR response responsible for effectively buffering proteotoxic stress. Our findings provide evidence for a role of metformin in ALL therapy and support strategies targeting synthetic lethal interactions with Akt and PIM kinases as suitable for future consideration for clinical translation in ALL

Abstract 4244: Novel expressed long non-coding RNAs in uveal melanoma

Abstract Uveal melanoma (UM) is a highly aggressive eye cancer that leads to metastatic death in up to half of patients. UMs can be divided into two prognostic groups based on their gene expression profile (GEP). The class 1 GEP is associated with low metastatic risk and the class 2 GEP with high metastatic risk. Class 1 tumors are associated with EIF1AX or SF3B1 mutations while Class 2 tumors are associated with inactivating mutations in the tumor suppressor BAP1. Class 1 and Class 2 UMs have been shown to differ in their expression of numerous known micro-RNAs and long non-coding RNAs (lncRNA). Here, we sought to identify novel differentially expressed lncRNAs using a publicly available RNA-Seq database. Raw RNA-Seq fastq files from 80 TCGA UM samples were obtained from the Cancer Genomics Hub (CGHub), quality controlled using FastQC (v0.11.3), and trimmed using trim-galor (v0.4.1). Sequences were aligned to the human genome (GRCh38) and accompanying general transfer format file (gtf) (Gencode v28) using STAR (v2.5). Transcript discovery was performed using Cufflinks (v2.2.1). Protein coding probability was calculated using CPC (v2.0), and transcripts predicted to be non-coding with transcript length >200bps were retained. 1671 novel transcripts were added to the gtf file, and fastq files were realigned with the new annotation. Estimated counts for all known and novel transcripts were generated using RSEM (v1.3.0) after STAR alignment. A cutoff of RPKM > 1 in at least 35% of tumors was used as a threshold for transcripts of interest, resulting in 61 novel transcripts, 32 of which were differentially expressed at FDR < 0.05 between Class 1 and Class 2 tumors, including 7 upregulated and 25 downregulated in Class 2 tumors. Further work is underway to elucidate the function of these novel transcripts in UM pathogenesis. Citation Format: Daniel A. Rodriguez, Jeffim N. Kuznetsov, Margaret I. Sanchez, Stefan Kurtenbach, J. William Harbour. Novel expressed long non-coding RNAs in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4244

AMPK and Akt Determine Apoptotic Cell Death following Perturbations of One-Carbon Metabolism by Regulating ER Stress in Acute Lymphoblastic Leukemia

AICAr is a cell-permeable nucleotide that has been used in vivo and in vitro to activate AMPK. Our previous findings have shown that AICAr as a single agent induces dose- and time-dependent growth inhibition in acute lymphoblastic leukemia (ALL) cell lines. In addition, the combination of AICAr with antifolates (methotrexate (MTX) or pemetrexed) has been shown to further potentiate AMPK activation and to lead to greater cytotoxicity and growth inhibition in leukemia and other malignant cell types. Our data presented herein demonstrate that sustained ER stress is the predominant mechanism behind the synergistic induction of cell death by the combination of AICAr plus the inhibitor of one-carbon metabolism, MTX, in Bp- and T-ALL, as evidenced by induction of several unfolded protein response markers leading to apoptosis. We also show for the first time that AICAr in combination with MTX significantly induces Akt phosphorylation in ALL. Under these conditions, the concomitant inhibition of Akt, a cellular antagonist of AMPK, leads to further up-regulation of AMPK activity and alleviates AICAr plus MTX-induced ER stress and apoptosis. Therefore, we also demonstrate that the concomitant activation of AMPK actually rescues the cells from AICAr plus MTX-induced ER stress and apoptosis. Our data suggest that the effects of AMPK activation on cell death or survival differ contextually depending on its signaling alterations with related oncogenic pathways and provide insight into the reported paradoxical pro-apoptotic vs. pro-survival effects of AMPK activation

Abstract 1541: The tumor suppressor BAP1 promotes a developmental switch from pluripotency to differentiation

Abstract Introduction: Our lab discovered that mutations in the tumor suppressor BAP1 are strongly associated with metastasis and death in patients with uveal melanoma. Subsequently, other cancers have been found to harbor BAP1 mutations, including skin melanoma, kidney cancer, mesothelioma and others. Germline BAP1 mutations are responsible for a newly described genetic cancer syndrome. Therapeutic molecules that reverse the effects of BAP1 mutations could represent a potent new treatment strategy for BAP1-mutant cancers. Unfortunately, there are several obstacles to developing such therapies. First, BAP1 is a tumor suppressor that is inactivated by mutations, such that targeted therapy would need to be directed against downstream effectors that are deregulated by BAP1 loss. Second, the effectors of BAP1 that are relevant to cancer are not known. Interestingly, most known proteins that interact with Bap1 are developmental epigenetic regulators such as Asxl1/2, Cbx1/3 and Kdm1b. Third, BAP1 is difficult to study in cultured cells because BAP1 loss results in cell cycle exit and stem cell-like behavior. These obstacles led us to shift to Xenopus laevis as an in vivo developmental model to study the functions of BAP1. Results and conclusions: Loss of BAP1 during embryo development results in a failure to turn off pluripotency genes such as ventx 1, ventx2 (Xenopus orthologues of the mammal gene nanog), oct 25, oct 91 (Xenopus orthologues of the mammal gene oct3/4) and pax3, and a failure to induce lineage specification genes such as the prospective epidermis marker keratin1 and the melanocyte precursor marker sox10. This block in the shift from pluripotency to differentiation programs results in a delay in gastrulation, neural crest specification and migration, mesodermal differentiation and other phenotypes. The BAP1-deficient phenotype can be rescued by Xenopus or human wildtype BAP1 or by the histone deacetylase inhibitor SAHA (vorinostat). We conclude that BAP1 is a fundamental regulator of multiple developmental lineages, including the neural crest from which melanomas arises, and that this in vivo model can be used to screen for novel therapeutic compounds that reverse the phenotypic effects of BAP1 loss. Citation Format: Jeffim N. Kuznetsov, Tristan Aguero, Stefan Kurtenbach, Matthew G. Field, Mary Lou King, J William Harbour. The tumor suppressor BAP1 promotes a developmental switch from pluripotency to differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1541. doi:10.1158/1538-7445.AM2017-1541</jats:p