Acquisition Of Therapy Resistance Research Articles

Abstract 540: Identification of therapy resistance mediating extracellular vesicles in single-cell level via a novel microfluidic co-culture device development

Abstract Once cancer has metastasized, it is incurable because tumors evolve resistance to all systemically administered anti-cancer therapies. Resistance to therapy has classically been attributed to the presence of a cancer stem cell or genetic tumor cell heterogeneity. Extracellular vesicles (EVs) are nano-sized (40-1000 nm) vesicles that are released by all cells into the extracellular space. EVs carry diverse cargo dependent on the cell of origin and have been studied as a source of novel cancer-specific liquid biopsy biomarkers and contributors to tumor progression and metastasis. However, the paracrine function of EVs in promoting therapy resistance is under-studied, and the impact of EVs released from therapy-resistant cancer cells on surrounding therapy-sensitive cancer cells is unknown. In this study, we develop an innovative microfluidic device, TREVCo, to enable simultaneous single-cell trap, EV detection, and co-culture modules to evaluate EVs-mediated transfer of cellular information between therapy-resistant cancer cells and adjacent cells, and to characterize EVs released from resistant cells and determine their impact on the resistance phenotype of adjacent therapy-sensitive cancer cells. We utilize resistant cells from multiple prostate (PCa) cell lines that have been selected for therapy resistance by culture with increasing dosages of cancer drugs such as cisplatin, docetaxel. We use the matched untreated therapy-sensitive parental cell lines as the sensitive cells. We hypothesize a novel model of therapy resistance in lethal PCa: EVs released from resistant cells contain pro-resistance factors that are transferred to adjacent sensitive cells upon EV uptake, thus directly enabling therapeutic resistance. We will assess the impact of resistant cell-EVs at a single cell level via a novel device, TREVCo, that enables comprehensive downstream analysis. This single-cell approach enables dissection of critical EV-associated therapy-resistant factors within the large heterogeneity of resistant PCa. Co-culture of a single resistant cell and a hundred adjacent sensitive cells mimicking intra-tumoral interactions is also critical to demonstrate evidence of resistant cell secreting factors inducing a pro-resistance phenotype. These studies will be the first-in-field to assess the impact of resistant PCa cell-derived EVs at single-cell resolution. An understanding of resistant cell-derived EVs and the impact of that EV cargo on adjacent sensitive cells will provide critical insight into the acquisition of therapy resistance in lethal PCa. The novel approach to apply resistant cell-derived EVs in co-culture assay with sensitive cells will enable the screening of transmittable resistance-associated factors and provides critical opportunities for accelerating precision medicine for advanced PCa patients. Citation Format: Chi-Ju Kim, Sarah Amend, Kenneth Pienta. Identification of therapy resistance mediating extracellular vesicles in single-cell level via a novel microfluidic co-culture device development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 540.

Acquisition of 1pLOH-1qGain Is a Frequent Trajectory in Expanding Relapsed/Refractory Myeloma Subclones

Genetic clonal heterogeneity is common in multiple myeloma (MM). However, the specific drivers of subclonal evolution associated with disease progression under therapeutic selection has not been fully characterized with the resource of sequential samples to accurately observe changes in relation to drug exposures. To address this gap, we generated whole-genome sequencing data for 2-4 sequential samples of 50 UK relapsed/refractory MM (rrMM) patients taken before, during and after acquisition of therapy resistance. These patients were exposed to a real-world range of multi-agent therapy combinations between 2011 and 2020 all of which included IMiDs. We undertook high-resolution subclonal heterogeneity analysis using Battenberg and multi-dimensional DPClust as gold-standard methods [Salcedo et al. 2020, PMID: 31919445] and implemented the Plackett-Luce model of partial rankings to time genomic events based on their cancer cell fraction and identify alternative evolutionary trajectories [Ansari-Pour et al. 2021, PMID: 34836952]. This approach enabled the identification and tracking of recurrent genomic aberrations gaining dominance amongst the evolving subclones of the disease in relation to therapeutic exposure and resistance acquisition, and to our knowledge is the first time that has been applied to analyze multi-timepoint myeloma WGS datasets. We observed recurrent selection of subclones harbouring 17p loss of heterozygosity (LOH) or 1pLOH with disease progression and therapy resistance acquisition in tumour phylogenies. In addition, clonal timing analysis of the longitudinal dataset suggested that sequential acquisition of 1pLOH and 1qGain in the same clone was a common selected trajectory (22% of cases). To validate this finding, the same trajectory analysis was undertaken in a much larger IMiD-refractory rrMM WGS as a validation dataset (N=386, Myeloma Genome Project), albeit with single time-point samples and compared with a set of unrelated newly diagnosed WGS dataset (N=198, IFM 2009) [Ansari-Pour et al. 2023, PMID: 36223594]. Strikingly, 1pLOH was found to be the only event that showed evidence for clonal selection in this case-control analysis (timing rank fold-change = 3.7, P<2.2x10 -16). Interestingly, the 1pLOH-1qGain trajectory (27% of cases; co-occurrence OR = 2.3 (95% CI 1.5-3.8), P=2x10 -4) was also identified in the validation dataset, corroborating the potential driverness of the dual chromosome 1 copy number events in subclonal expansion during therapy resistance acquisition. While this WGS-based analysis of selected evolutionary trajectories in relapsed/refractory myeloma patient tumours needs further confirmation with sequential WGS datasets from uniformly treated cohorts, these findings suggest potential collaborative genetic interaction between 1pLOH and 1qGain in the same cell as emerging co-drivers of resistance/disease progression, conferring a greater selective advantage under therapeutic exposure than either event on its own. If confirmed, these biomarkers may define a new ‘double hit’ in relapsed/refractory myeloma, with biological consequences that will require novel and directed therapeutic approaches.

Abstract P2-23-19: Interrogating multicellular signaling in breast cancer using a bio-orthogonal chemistry-based proteomics platform

Abstract Tumors are multicellular ecosystems that communicate through the exchange of extracellular signaling molecules. In breast cancer, this multicellular communication can enable tumor cells to cooperate in a range of contexts, including tumor invasion and the outgrowth of distal metastases. Illuminating the mechanisms by which tumor cells communicate may reveal new therapies. To this end, three-dimensional tumor organoid models have emerged as versatile platforms for modeling multicellular behavior ex vivo. However, organoid culture typically requires the use of poorly defined, animal-derived extracellular matrices, such as Matrigel. These exogenous matrices contain thousands of proteins that dominate over and conceal cell-secreted factors in conventional proteomics approaches. Revealing low abundance cell-secreted factors from this complex exogenous background presents a formidable challenge. To surmount this challenge, we develop a new method to isolate the pericellular proteome in 3D organotypic culture models. This technology harnesses biorthogonal click chemistry to bypass exogenous factors, infiltrate intercellular spaces, and retrieve the endogenous intercellular proteome. This approach requires no genetic manipulation, requires only 1 million cells, and can be adapted to diverse organotypic models. These capabilities enable isolation of intercellular signaling factors in models that yield precious amounts of material and that can sustain only limited manipulation ex vivo, which we demonstrate using organoids established from patient-derived xenografts. To establish the generalizability of our approach, we apply this technology to a panel of breast cancer and small-cell lung cancer organoid models. Furthermore, we demonstrate that this method can be readily adapted to different extracellular matrix environments that are widely employed in organoid research, including basement membrane matrices and collagen gels. Taken together, our results establish this technology as a scalable and generalizable platform that may open opportunities for researchers investigating diverse questions in organoid models of cancer. Moving forward, we are leveraging these capabilities to investigate changes in the intercellular proteome that emerge after the acquisition of therapy resistance. We hope to uncover new mechanisms of collective signaling that may be targeted to overcome therapy resistance. Citation Format: Brad A. Krajina, Ami Yamamoto, Kevin J. Cheung, Samuel Madasu. Interrogating multicellular signaling in breast cancer using a bio-orthogonal chemistry-based proteomics platform [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-23-19.

Precision Medicine Approaches to Overcome Resistance to Therapy in Head and Neck Cancers.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most incident cancer worldwide. More than half of HNSCC patients experience locoregional or distant relapse to treatment despite aggressive multimodal therapeutic approaches that include surgical resection, radiation therapy, and adjuvant chemotherapy. Before the arrival of immunotherapy, systemic chemotherapy was previously employed as the standard first-line protocol with an association of cisplatin or carboplatin plus 5-fluorouracil plus cetuximab (anti-EFGR antibody). Unfortunately, acquisition of therapy resistance is common in patients with HNSCC and often results in local and distant failure. Despite our better understanding of HNSCC biology, no other molecular-targeted agent has been approved for HNSCC. In this review, we outline the mechanisms of resistance to the therapeutic strategies currently used in HNSCC, discuss combination treatment strategies to overcome them, and summarize the therapeutic regimens that are presently being evaluated in early- and late-phase clinical trials.

Abstract ES4-1: Adaptive Resistance to Hormonal Therapy

Abstract While recurrences are delayed by the use of endocrine therapy (ET), resistance can evolve with the development of metastatic breast cancer (MBC) in about one-fourth of ER-positive patients. MBC is incurable with a median 5-year survival rate of less than 25%. We know that ET resistance can result from an adaptive process in which escape survival mechanisms are used to evade treatment. One of the best-characterized ET resistance mechanisms is transcriptional activation of growth factor receptor (GFR) signaling, and subsequent estrogen-independent activation of ER via “cross-talk” with downstream signaling from GFRs. We will discuss distinct mechanisms of ligand-independent activation of ER discovered using transcriptional profiling of metastatic, ET-resistant tumors. It is now estimated that ~20% of therapy-resistant MBCs contain ESR1 mutations. Recent data has also shown that mutations in the MAPK/RAS pathway and transcription factor regulatory programs (for example, MYC) only account for an additional 22% of recurrent breast tumors. Thus, acquired mutations alone do not account for the majority of ET resistance mechanisms in MBC. ER-positive breast cancer cell lines display profound transcriptional variability at the single cell level associated with the acquisition of therapy resistance. Recently, bulk and single cell RNA sequencing was used on sequential samples from patients with ER-positive, ET-resistant patients and identified RNA expression patterns that represented different cellular phenotypes, including an increased epithelial mesenchymal transition state that could provide a selective advantage for selective phenotypic subclones to dominate. We will focus our discussion of transcriptional reprograming as a major mechanism of ET resistance. Citation Format: S Fuqua. Adaptive Resistance to Hormonal Therapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr ES4-1.

Abstract 349: Oncolytic reovirus variants induce direct oncolysis in human prostate cancer

Abstract The acquisition of therapy resistance and the formation of distant metastases represent major problems in prostate cancer. To date, immunotherapeutic approaches have been relatively disappointing in the treatment of prostate cancer. Therapies based on oncolytic viruses are being exploited for several tumor entities and have been studied in multiple clinical trials. Mammalian orthoreovirus is a non-enveloped double stranded RNA virus that has not been linked to serious disease in humans. Reovirus is relatively safe to use and several clinical trials are currently ongoing to study the oncolytic effect of reovirus in various tumor types. Type 3 Dearing (T3D) is a promising candidate for the use as an oncolytic agent. Besides wildtype T3D reovirus (R124), spontaneous mutants Jin1, 2 and 3 were generated which harbor mutations in spike protein Sigma-1. In this way, Jin mutants are able to infect cells independently of the Junction Adhesion Molecule-A (JAM-A) entry receptor on the tumor cell surface, which is often downregulated in different types of cancer. In this study, the direct oncolytic effect of wildtype (R124) and Jin mutant reovirus was examined in human prostate cancer cells in vitro and in our ‘near-patient’ model of ex vivo cultured human prostate cancer tissue slices. Both R124 and Jin3 dose-dependently induced prostate cancer cell death in PC-3M-Pro4luc2, DU145 and 22Rv1 cells in vitro. Interestingly, JAM-A negative cancer cells were insensitive for R124, but were effectively killed by Jin1 mutant, indicating the beneficial effects of the Jin mutant in JAM-A low/negative tumors. Moreover, tumor tissue slices were generated from explanted patient prostate cancer tissues and patient-derived xenografts (PDX) and cultured with reovirus. Oncolytic effects were observed in ex vivo cultured tumor slices upon viral replication and subsequent stimulation of apoptosis. Therefore, ex vivo culture of patient-derived tumor tissue allows the assessment of individual anti-tumor response of the oncolytic virotherapy (i.e. personalized therapeutics). Taken together, our results indicate that both wildtype R124 virus and mutant Jin reovirus can infect and replicate in human prostate cancer cells and patient-derived prostate cancer tissues. The Jin mutants are of particular interest in JAM-A negative tumors. The indirect immunomodulatory effects of reovirus in human prostate cancer are currently ongoing. In conclusion, our selected oncolytic reoviruses may represent a promising novel treatment strategy in human prostate cancer alone or combined with immunostimulatory agents. Citation Format: Arjanneke F. van de Merbel, Geertje van der Horst, Maaike H. van der Mark, Diana J. van den Wollenberg, Jack A. Schalken, Anne Collins, Norman J. Maitland, Rob C. Hoeben, Gabri van der Pluijm. Oncolytic reovirus variants induce direct oncolysis in human prostate cancer [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 349.

MYC Expression and Metabolic Redox Changes in Cancer Cells: A Synergy Able to Induce Chemoresistance.

Chemoresistance is due to multiple factors including the induction of a metabolic adaptation of tumor cells. In fact, in these cells, stress conditions induced by therapies stimulate a metabolic reprogramming which involves the strengthening of various pathways such as glycolysis, glutaminolysis and the pentose phosphate pathway. This metabolic reprogramming is the result of a complex network of mechanisms that, through the activation of oncogenes (i.e., MYC, HIF1, and PI3K) or the downregulation of tumor suppressors (i.e., TP53), induces an increased expression of glucose and/or glutamine transporters and of glycolytic enzymes. Therefore, in order to overcome chemoresistance, it is necessary to develop combined therapies which are able to selectively and simultaneously act on the multiple molecular targets responsible for this adaptation. This review is focused on highlighting the role of MYC in modulating the epigenetic redox changes which are crucial in the acquisition of therapy resistance.

Abstract 898: Clear cell renal cell carcinoma resistance to RTKs inhibitors is mediated by c-Met receptor and MCPIP1

Abstract Introduction Clear cell renal cell carcinoma (ccRCC) treatment with small molecules that inhibit multiple receptor tyrosine kinases (RTKs),showed in approximately 38% of patients significant tumor control. However, despite the efficacy of treatment, ccRCC often develops resistance to targeted drugs and the majority of patients who receive such treatment exhibit progressive disease after 1 year. Several hypotheses have been proposed regarding the mechanisms underlying resistance to RTKs inhibitors, but the precise pathways have not yet been fully elucidated. Aim The main objective of our study was to determine the role of c-Met and anti-inflammatory protein MCPIP1 in the acquisition of resistance to RTKs inhibitors in ccRCC. Materials and methods ccRCC cell lines (Caki-1 and Caki-2) as well as normal epithelial cell lines (Hek293, RPTEC/TERT1) were treated with sunitinib, sorafenib or SU11274 constantly for 24h, 1 and 3 weeks. The level and localization of key proteins were examined using western blots and IF staining. ccRCC patient samples were divided into four histological grades, and levels of genes or proteins were investigated using microarray analysis, western blots, protein arrays, mass spectrometry and real time PCR. Results Our study shows that short term (24h) and long term (3 weeks) stimulation with sunitinib or sorafenib induced phosphorylation of c-Met receptor, STAT3 and Src kinase. Furthermore, sunitinib treatment resulted in the acquisition of cancer stem cells features and the formation of clones. In addition, after sunitinib and sorafenib treatment we observed a decrease in the protein level of MCPIP1, postulated tumor suppressor. Interestingly, after MCPIP1 overexpression, we observed c-Met downregulation, regulated by RNase activity of MCPIP1. Moreover, our results show that in patient samples, together with MCPIP1 downregulation, level of phosphorylated and total c-Met receptor increase. Conclusions We showed that acquisition of therapy resistance in ccRCC may be affected by MCPIP1 decrease, together with phopsphorylation of c-Met receptor and it can be partially reversed by overexpressing the MCPIP1 protein, which may act as a potent tumor suppressor. Proposed research may help in understanding the mechanisms responsible for tumor resistance to targeted therapy. Obtained results may contribute to increased understanding of the biology of clear cell renal cell carcinoma, which in the future may help in identifying new, more effective therapeutic purposes or improving existing ones. Acknowledgment This study was supported by research grant from the National Science Centre 2013/09/D/NZ/00249, 2017/25/N/NZ5/03014 and grant from Jagiellonian University BMN 16/2017. Citation Format: Paulina Marona, Judyta Górka, Jolanta Jura, Katarzyna Miękus. Clear cell renal cell carcinoma resistance to RTKs inhibitors is mediated by c-Met receptor and MCPIP1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 898.

Abstract 2433: The hypoxic tumor microenvironment in vivo selects the tumor cells with cancer stem cell characteristics and increased resistance against genotoxic stresses

Abstract Tumor hypoxia has been established as an independent prognostic factor associated with poor patient survival and resistance to conventional therapies. Emerging evidence suggests that hypoxia has the potential to maintain or enhance the stem cell phenotype and confers chemo- or radio-resistance to tumor cells. However, it remains to be determined whether cell fate and acquisition of therapy resistance is regulated in vivo by the hypoxic tumor microenvironment (TME). In this study, we have developed a hypoxia-sensing human breast cancer cell xenograft model using the hypoxia-response element (HRE)-driven enhanced green fluorescence protein (EGFP) as a hypoxia reporter. We have found that the hypoxic tumor cells isolated from xenografts contain increased subpopulations of tumor cells showing the cancer stem cell (CSC) characteristics. Upon re-implantation, the CSC characteristics of the hypoxic tumor cells continue to be further enriched during in vivo growth, whereas secondary xenografts derived from the non-hypoxic tumor cells remain similar to the primary xenografts. Consistent with this finding, the phenotypes exhibited by the hypoxic tumor cells are stably different from those of the non-hypoxic tumor cells isolated from the same tumor mass even when they are maintained under the same ambient culture conditions. Mechanistically, we find that the PI3K/AKT pathway is strongly potentiated in the hypoxic tumor cells and is required to maintain the CSC phenotype. Cell fate differences between hypoxic and non-hypoxic tumor cells are found only in tumor cells isolated from the hypoxic TME in vivo, and are not seen in tumor cells treated by hypoxia in vitro alone. In addition, we have found that naturally hypoxic tumor cells in a breast cancer xenograft model are moderately radio- and chemo-resistant compared to their neighboring non-hypoxic tumor cells in the same tumor. These naturally hypoxic tumor cells are proficient at repairing DNA damages and resist apoptosis induced by genotoxic stresses. The ex vivo hypoxic tumor cells are capable of proficient activation of the DNA damage-sensing pathways, especially the ATM pathway. These previously unrecognized observations suggest that the hypoxic TME may promote malignant progression by coordinating induction, selection and preferential maintenance of the CSC phenotype in tumor cells. This presentation will review our results which strongly suggest that the hypoxic TME may induce adaptive responses, either by clonal evolution or selection, such that hypoxic tumor cells in vivo acquire new functional traits that confer resistance to genotoxic stresses and enhance survival. It can be postulated that previously hypoxic tumor cells may continue to exhibit their aggressive phenotypes long after they have migrated into non-hypoxic regions or after their native niche become normoxic. Citation Format: Hoon Kim, Qun Lin, Zhong Yun. The hypoxic tumor microenvironment in vivo selects the tumor cells with cancer stem cell characteristics and increased resistance against genotoxic stresses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2433.

Abstract 513: The link between polyploidy and replication stress in melanoma

Abstract Genomic instability is a hallmark of cancer implicated in tumor evolution and resistance to therapy. However, the molecular mechanisms that underlie genomic instability are still poorly understood. One common feature of cancer cells is an elevated genomic content (polyploidy) which is also associated with the acquisition of therapy resistance. Here we demonstrate a link between genomic instability and polyploidization in melanoma cells. We show that polyploidy induced by treatment with small molecule inhibitors of various mitotic kinases, such as AURKA, AURKB and PLK1, leads to DNA damage in cells with elevated (>4n) DNA content. This DNA damage results from replication stress exacerbated by limited activation of p53 in malignant melanocytes. Pharmacological induction of p53 in polyploid cells using an MDM2 antagonist reduced DNA damage by blocking re-replication of the polyploid genome as a result of p53-mediated p21 activation and transactivation of DNA repair genes. Notably we found that p21 blockade using siRNA knockdown or genetic knockout shifted polypoid cell response to p53 activation from cytostatic to cytotoxic. As a result, p21-deficient cells exhibited enhanced sensitivity to mitotic blockade combined with p53 induction. Furthermore, TCGA dataset analysis showed poor progression free survival in melanoma patients with high p21 protein expression. These data argue for administering mitotic inhibitors and MDM2 antagonists, which are currently in clinical development, in conjunction with agents that target p21. In summary, our data here reveal that polyploidization can be a mechanism for induction of DNA damage and genomic instability associated with drug resistance in cancer and suggest a novel strategy for targeting these pathways to improve melanoma therapy. Citation Format: Anna E. Vilgelm, C. Andrew Johnson, Kiran Malikayil, Dayanidhi Raman, David Flaherty, Brian Higgins, Ann Richmond. The link between polyploidy and replication stress in melanoma [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 513. doi:10.1158/1538-7445.AM2017-513

Nrf2 promotes mutant K-ras/p53-driven pancreatic carcinogenesis.

The Keap1-Nrf2 system contributes to the maintenance of homeostasis by regulating oxidative stress responses in normal tissues and organs, and is exploited in various cancers for proliferation, survival and acquisition of therapy resistance. Pancreatic cancer remains one of the intractable cancers, despite the improved clinical outcomes of other types of cancer, due to its invasive and refractory nature to therapeutic intervention. The current study aimed to clarify the contribution of Nrf2 to pancreatic carcinogenesis using a pancreas-specific mutant K-ras and p53 (KPC) mouse model. Deletion of Nrf2 in KPC mice (KPCN) decreased the formation of precancerous lesions as well as the development of invasive pancreatic cancer. The pancreatic tumor-derived cancer cell lines from KPCN mouse showed decreased expression of glutathione S-transferases (GST), UDP glucuronosyltransferases (UGT) and ABC transporters. Along with these biochemical changes, cell lines from KPCN mice revealed increased sensitivity to oxidative stress and chemotherapeutic agent. The current study revealed that Nrf2 contributes to pancreatic carcinogenesis in a way distinct from the chemoresistance of lung and esophagus, and that Nrf2 could be a novel therapeutic target of pancreatic cancer.

Mechanisms of Therapy Resistance in Patient-Derived Xenograft Models of BRCA1-Deficient Breast Cancer

Although BRCA1-deficient tumors are extremely sensitive to DNA-damaging drugs and poly(ADP-ribose) polymerase (PARP) inhibitors, recurrences do occur and, consequently, resistance to therapy remains a serious clinical problem. To study the underlying mechanisms, we induced therapy resistance in patient-derived xenograft (PDX) models of BRCA1-mutated and BRCA1-methylated triple-negative breast cancer. A cohort of 75 mice carrying BRCA1-deficient breast PDX tumors was treated with cisplatin, melphalan, nimustine, or olaparib, and treatment sensitivity was determined. In tumors that acquired therapy resistance, BRCA1 expression was investigated using quantitative real-time polymerase chain reaction and immunoblotting. Next-generation sequencing, methylation-specific multiplex ligation-dependent probe amplification (MLPA) and Target Locus Amplification (TLA)-based sequencing were used to determine mechanisms of BRCA1 re-expression in therapy-resistant tumors. BRCA1 protein was not detected in therapy-sensitive tumors but was found in 31 out of 42 resistant cases. Apart from previously described mechanisms involving BRCA1-intragenic deletions and loss of BRCA1 promoter hypermethylation, a novel resistance mechanism was identified in four out of seven BRCA1-methylated PDX tumors that re-expressed BRCA1 but retained BRCA1 promoter hypermethylation. In these tumors, we found de novo gene fusions that placed BRCA1 under the transcriptional control of a heterologous promoter, resulting in re-expression of BRCA1 and acquisition of therapy resistance. In addition to previously described clinically relevant resistance mechanisms in BRCA1-deficient tumors, we describe a novel resistance mechanism in BRCA1-methylated PDX tumors involving de novo rearrangements at the BRCA1 locus, demonstrating that BRCA1-methylated breast cancers may acquire therapy resistance via both epigenetic and genetic mechanisms.

미세소관-손상 항암제 처리에 의한 세포주기의 정지 및 에폽토시스 유도

에폽토시스에 의한 세포자멸사는 암세포에 대한 항암제 효능의 핵심적 기전이다. 항암제의 대표적인 두 종류로 알려진 DNA-손상 약제(DNA-damaging agents, DDAs)와 미세소관-손상 약제(microtubule-damaging agents, MDAs)가 암세포에 야기하는 초기 항암신호전달 기전은 다르지만, 최종적으로는 대부분 미토콘드리아 의존-에폽토시스를 통해 암세포를 사멸시킨다. 한편, DDAs에 의한 에폽토시스 유도에는 wild-type 종양억제 단백질 p53의 역할이 매우 중요하다. 그러나 인체 암의 약 50% 이상이 p53유전자의 돌연변이 때문에 종양억제 단백질로서의 p53 기능이 불활성화 되어 있다. 따라서 p53과 무관하게 에폽토시스를 유도할 수 있는 MDAs를 이용한 항암치료는 돌연변이 p53을 지닌 암세포에 대해 유리한 화학요법으로 이해된다. 최근 본 연구진은 인체 급성 백혈병 세포주인 Jurkat T 세포를 모델로 하여, MDAs (nocodazole, 17-α-estradiol, 혹은 2-methoxyestradiol)의 항암작용과 관련된 세포주기 정지 및 에폽토시스 유도 기전을 구명하였다. 그 결과, Jurkat T 세포를 MDAs로 처리할 경우, 유사분열방추사의 결함에 의한 세포주기(전중기, prometaphase) 정지, 장시간에 걸친 Cdk1의 활성화, 활성화된 Cdk1에 의한 에폽토시스 조절인자들(Bcl-2, Bcl-xL, Mcl-1 및 Bim)의 인산화, 이에 따른 Bak 활성화, 미토콘드리아막 손상 및 카스파아제 연쇄 활성화에 의해 에폽토시스가 유도됨을 밝혔다. 또한 동일한 MDA 처리 조건하에서 Bcl-2 혹은 Bcl-xL의 과발현시켜 에폽토시스 진행을 차단할 경우, Jurkat T 세포는 약제처리 후에 전중기 정지된 4N 상태에 도달하지만, 이어서 유사분열 불이행(mitotic slippage) 및 내재복제(endoreduplication)가 진행되어 다배수체들(polyploids; 8N, 16N)을 생성하게 됨을 확인하였다. 이러한 결과는 MDAs처리에 따른 다배수체들의 생성을 차단하는 세포 내 기전으로서, 전중기 정지된 4N 세포의 에폽토시스에 의한 제거가 매우 중요함을 보여준다. 특히, 다배수체는 유전적으로 매우 불안정하여 암세포의 항암제 내성 획득 및 암 재발과 직접 연관되는 것으로 알려져 있으므로, 에폽토시스 기전에 결함이 있는 암세포를 대상으로 MDAs를 이용한 항암 화학요법을 시행할 경우에는 다배수체 세포의 생성을 차단하기 위한 새로운 수단이 반드시 병행되어야 할 것으로 사료된다. Apoptosis induction has been proposed as an efficient mechanism by which malignant tumor cells can be removed following chemotherapy. The intrinsic mitochondria-dependent apoptotic pathway is frequently implicated in chemotherapy-induced tumor cell apoptosis. Since DNA-damaging agent (DDA)-induced apoptosis is mainly regulated by the tumor suppressor protein p53, and since more than half of clinical cancers possess inactive p53 mutants, microtubule-damaging agents (MDAs), of which apoptotic effect is mainly exerted via p53-independent routes, can be promising choice for cancer chemotherapy. Recently, we found that the apoptotic signaling pathway induced by MDAs (nocodazole, 17α-estradiol, or 2-methoxyestradiol) commonly proceeded through mitotic spindle defect-mediated prometaphase arrest, prolonged Cdk1 activation, and subsequent phosphorylation of Bcl-2, Mcl-1, and Bim in human acute leukemia Jurkat T cells. These microtubule damage-mediated alterations could render the cellular context susceptible to the onset of mitochondria-dependent apoptosis by triggering Bak activation, Δψm loss, and resultant caspase cascade activation. In contrast, when the MDA-induced Bak activation was inhibited by overexpression of anti-apoptotic Bcl-2 family proteins (Bcl-2 or Bcl-xL), the cells in prometaphase arrest failed to induce apoptosis, and instead underwent mitotic slippage and endoreduplication cycle, leading to formation of populations with 8N and 16N DNA content. These data indicate that cellular apoptogenic mechanism is critical for preventing polyploid formation following MDA treatment. Since the formation of polyploid cells, which are genetically unstable, may cause acquisition of therapy resistance and disease relapse, there is a growing interest in developing new combination chemotherapies to prevent polyploidization in tumors after MDA treatment.

Abstract IA04: Neuroblastoma as a paradigm of MYC(N) driven cancers

Abstract Neuroblastoma is an enigmatic childhood cancer with phenotypes ranging from spontaneous regression of seemingly advanced disease to relentless metastatic progression. MYCN amplification was defined as a powerful predictor of metastatic potential and acquisition of therapy resistance in the 1980's, and arguably was the first genomic biomarker utilized in routine clinical practice to stratify therapeutic intensity. While there have been incremental improvements in cure rates over the last three decades, these have come at the cost of a highly complex and intensive chemoradiotherapeutic regimen that leaves survivors with significant long-term morbidity. Here we will discuss our ongoing efforts to define the genetic basis of neuroblastoma, and recent insight into how heritable DNA variation influences neuroblastoma susceptibility and clinical phenotypes, including MYCN amplification. We will also discuss the major oncogenic drivers of high-risk neuroblastoma beyond MYCN, including recent insights into the role of MYC in the MYCN-non-amplified high-risk cases, as well as the major role of the ALK oncogene, often in cooperation with MYCN. Finally, we will discuss recent efforts to leverage this information therapeutically, with a focus on how therapeutically targetable oncogenic pathways evolve under the selective pressure of chemoradiotherapy. Finally, we will discuss nascent efforts to deliver on the holy grail of neuroblastoma translational research—targeting the MYCN oncoprotein itself, discussing opportunities and challenges. Citation Format: John M. Maris. Neuroblastoma as a paradigm of MYC(N) driven cancers. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr IA04.

Size Does Matter: Why Polyploid Tumor Cells are Critical Drug Targets in the War on Cancer.

Tumor evolution presents a formidable obstacle that currently prevents the development of truly curative treatments for cancer. In this perspective, we advocate for the hypothesis that tumor cells with significantly elevated genomic content (polyploid tumor cells) facilitate rapid tumor evolution and the acquisition of therapy resistance in multiple incurable cancers. We appeal to studies conducted in yeast, cancer models, and cancer patients, which all converge on the hypothesis that polyploidy enables large phenotypic leaps, providing access to many different therapy-resistant phenotypes. We develop a flow-cytometry based method for quantifying the prevalence of polyploid tumor cells, and show the frequency of these cells in patient tumors may be higher than is generally appreciated. We then present recent studies identifying promising new therapeutic strategies that could be used to specifically target polyploid tumor cells in cancer patients. We argue that these therapeutic approaches should be incorporated into new treatment strategies aimed at blocking tumor evolution by killing the highly evolvable, therapy-resistant polyploid cell subpopulations, thus helping to maintain patient tumors in a drug sensitive state.

Inhibition of the PI3K/Akt/GSK3 Pathway Downstream of BCR/ABL, Jak2-V617F, or FLT3-ITD Downregulates DNA Damage-Induced Chk1 Activation as Well as G2/M Arrest and Prominently Enhances Induction of Apoptosis

Constitutively-activated tyrosine kinase mutants, such as BCR/ABL, FLT3-ITD, and Jak2-V617F, play important roles in pathogenesis of hematopoietic malignancies and in acquisition of therapy resistance. We previously found that hematopoietic cytokines enhance activation of the checkpoint kinase Chk1 in DNA-damaged hematopoietic cells by inactivating GSK3 through the PI3K/Akt signaling pathway to inhibit apoptosis. Here we examine the possibility that the kinase mutants may also protect DNA-damaged cells by enhancing Chk1 activation. In cells expressing BCR/ABL, FLT3-ITD, or Jak2-V617F, etoposide induced a sustained activation of Chk1, thus leading to the G2/M arrest of cells. Inhibition of these kinases by their inhibitors, imatinib, sorafenib, or JakI-1, significantly abbreviated Chk1 activation, and drastically enhanced apoptosis induced by etoposide. The PI3K inhibitor GD-0941 or the Akt inhibitor MK-2206 showed similar effects with imatinib on etoposide-treated BCR/ABL-expressing cells, including those expressing the imatinib-resistant T315I mutant, while expression of the constitutively activated Akt1-myr mutant conferred resistance to the combined treatment of etoposide and imatinib. GSK3 inhibitors, including LiCl and SB216763, restored the sustained Chk1 activation and mitigated apoptosis in cells treated with etoposide and the inhibitors for aberrant kinases, PI3K, or Akt. These observations raise a possilibity that the aberrant kinases BCR/ABL, FLT3-ITD, and Jak2-V617F may prevent apoptosis induced by DNA-damaging chemotherapeutics, at least partly through enhancement of the Chk1-mediated G2/M checkpoint activation, by inactivating GSK3 through the PI3K/Akt signaling pathway. These results shed light on the molecular mechanisms for chemoresistance of hematological malignancies and provide a rationale for the combined treatment with chemotherapy and the tyrosine kinase or PI3K/Akt pathway inhibitors against these diseases.

Cetuximab in the Treatment of Colorectal Cancer

The role of epidermal growth factor receptor (EGFR)-driven signaling in different stages of colorectal carcinogenesis, as well in the acquisition of therapy resistance, has been established. Multiple strategies have been developed for the therapeutic targeting of EGFR. Cetuximab is a chimeric monoclonal antibody selective for EGFR with efficacy alone or in combination with irinotecan in the treatment of metastatic colorectal cancer patients, who have progressed to using irinotecan-containing chemotherapy. Cetuximab is well tolerated and does not exacerbate the toxicity of concomitant chemotherapy. Based on this data, the combination of cetuximab with standard chemotherapy regimens such as irinotecan/ 5-FU/folinic acid (FA) or oxaliplatin/5-FU/FA are currently being investigated in Phase III trials for chemotherapy-naive patients with metastatic colorectal cancer.