Mechanisms Of Tumor Cell Resistance Research Articles

Overcoming statin resistance in prostate cancer cells by targeting the 3-hydroxy-3-methylglutaryl-CoA-reductase

Prostate cancer is the most prevalent malignancy in men. While diagnostic and therapeutic interventions have substantially improved in recent years, disease relapse, treatment resistance, and metastasis remain significant contributors to prostate cancer-related mortality. Therefore, novel therapeutic approaches are needed. Statins are inhibitors of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate pathway which plays an essential role in cholesterol homeostasis. Numerous preclinical studies have provided evidence for the pleiotropic antitumor effects of statins. However, results from clinical studies remain controversial and have shown substantial benefits to even no effects on human malignancies including prostate cancer. Potential statin resistance mechanisms of tumor cells may account for such discrepancies. In our study, we treated human prostate cancer cell lines (PC3, C4–2B, DU-145, LNCaP) with simvastatin, atorvastatin, and rosuvastatin. PC3 cells demonstrated high statin sensitivity, resulting in a significant loss of vitality and clonogenic potential (up to – 70%; p < 0.001) along with an activation of caspases (up to 4-fold; p < 0.001). In contrast, C4–2B and DU-145 cells were statin-resistant. Statin treatment induced a restorative feedback in statin-resistant C4–2B and DU-145 cells through upregulation of the HMGCR gene and protein expression (up to 3-folds; p < 0.01) and its transcription factor sterol-regulatory element binding protein 2 (SREBP-2). This feedback was absent in PC3 cells. Blocking the feedback using HMGCR-specific small-interfering (si)RNA, the SREBP-2 activation inhibitor dipyridamole or the HMGCR degrader SR12813 abolished statin resistance in C4–2B and DU-145 and induced significant activation of caspases by statin treatment (up to 10-fold; p < 0.001). Consistently, long-term treatment with sublethal concentrations of simvastatin established a stable statin resistance of a PC3SIM subclone accompanied by a significant upregulation of both baseline as well as post-statin HMGCR protein (gene expression up to 70-fold; p < 0.001). Importantly, the statin-resistant phenotype of PC3SIM cells was reversible by HMGCR-specific siRNA and dipyridamole. Our investigations reveal a key role of a restorative feedback driven by the HMGCR/SREBP-2 axis in statin resistance mechanisms of prostate cancer cells.

The tumor microenvironment: a key player in multidrug resistance in cancer

Abstract Cancer is the second leading cause of death worldwide. Although multiple new cancer treatments have emerged in recent years, drug therapy, mainly comprising chemotherapy, targeted therapy, and immunotherapy, remains the most common approach. The multidrug resistance (MDR) of cancer cells to various treatments remains a challenge. Scientists have always focused on the acquired drug resistance mechanisms of tumor cells themselves. However, recent evidence shows that the tumor microenvironment (TME) plays a critical role in regulating tumor cell progression, metastasis, immune escape, and drug resistance. In the TME, interactions between cancer cells and non-malignant cells often modify the TME and facilitate drug resistance. Therefore, elucidating this complex interaction mechanism is essential for the development of effective treatments. This review focuses on the role of the TME in promoting chemoresistance in tumor cells through the following mechanisms: (i) inhibiting the immune clearance of tumor cells and facilitating immune escape responses; (ii) stimulating the release of soluble paracrine factors to enhance tumor survival and growth; (iii) promoting survival and altering drug delivery through metabolic reprogramming; (iv) obstructing drug absorption by inducing changes in stomatal cells and blood vessels surrounding the tumor; and (v) inducing the cancer stem cell phenotype. This review also addresses a clinical treatment strategy for targeting the TME, providing insights and a basis for reversing multidrug resistance.

Glycolysis-associated lncRNAs in cancer energy metabolism and immune microenvironment: a magic key.

The dependence of tumor cells on glycolysis provides essential energy and raw materials for their survival and growth. Recent research findings have indicated that long chain non-coding RNAs (LncRNAs) have a key regulatory function in the tumor glycolytic pathway and offer new opportunities for cancer therapy. LncRNAs are analogous to a regulatory key during glycolysis. In this paper, we review the mechanisms of LncRNA in the tumor glycolytic pathway and their potential therapeutic strategies, including current alterations in cancer-related energy metabolism with lncRNA mediating the expression of key enzymes, lactate production and transport, and the mechanism of interaction with transcription factors, miRNAs, and other molecules. Studies targeting LncRNA-regulated tumor glycolytic pathways also offer the possibility of developing new therapeutic strategies. By regulating LncRNA expression, the metabolic pathways of tumor cells can be interfered with to inhibit tumor growth and metastasis, thus affecting the immune and drug resistance mechanisms of tumor cells. In addition, lncRNAs have the capacity to function as molecular markers and target therapies, thereby contributing novel strategies and approaches to the field of personalized cancer therapy and prognosis evaluation. In conclusion, LncRNA, as key molecules regulating the tumor glycolysis pathway, reveals a new mechanism of abnormal metabolism in cancer cells. Future research will more thoroughly investigate the specific mechanisms of LncRNA glycolysis regulation and develop corresponding therapeutic strategies, thereby fostering new optimism for the realization of precision medicine.

The effect of DNA methyltransferase 3A suppression in progression of the resistance phenotype in breast cancer cells

Introduction. Rearrangement of molecular pathways and activation of bypass signaling determine the progression of tumor cell resistance to various drugs. Study of the common features of resistant formation mechanisms is essential for breast and other cancer beneficial treatments.Materials and methods. The present work was performed on estrogen receptor α ERα-positive (ERα – estrogen receptor α) McF-7 breast cancer cells, established sublines resistant to the mTOR inhibitor rapamycin or antiestrogen tamoxifen, and ERα-negative MDA-MB-231 breast cancer cells. Methods used include MTT test, transient transfection, immunoblotting, real-time polymerase chain reaction and methylation analysis by bisulfite pyrosequencing.Results. We have shown that the resistance of breast cancer cells to targeted and hormonal drugs is associated with the suppression of DNA methyltransferase 3A (DNMT3A) and respective changes in DNA methylation; DNMT3A knockdown results in the partial resistance to both drugs demonstrating the pivotal role of DNMT3A suppression in the progression of cell resistance.Conclusion. Totally, the results obtained highlight the possible mechanism of tumor cell resistance to targeting/hormonal drugs based on the deregulation of DNMTs expression and demonstrate direct connection between DNMT3A suppression and resistance progression.

Inhibition of epithelial-to-mesenchymal transition augments antitumor efficacy of nanotherapeutics in pancreatic ductal adenocarcinoma.

Intrinsic drug resistance mechanisms of tumor cells often reduce intracellular drug concentration to suboptimal levels. Epithelial-to-mesenchymal transition (EMT) is a pivotal process in tumor progression and metastasis that confers an aggressive phenotype as well as resistance to chemotherapeutics. Therefore, it is imperative to develop novel strategies and identify new targets to improve the overall efficacy of cancer treatment. We developed SN38 (active metabolite of irinotecan)-assembled glycol chitosan nanoparticles (cSN38) for the treatment of pancreatic ductal adenocarcinoma (PDAC). Furthermore, cSN38 and the TGF-β1 inhibitor LY364947 formed composite nanoparticles upon self-assembly (cSN38 + LY), which obviated the poor aqueous solubility of LY364947 and enhanced drug sensitivity. The therapeutic efficacy of cSN38 + LY nanotherapeutics was studied in vitro and in vivo using suitable models. The cSN38 nanoparticles exhibited an antitumor effect that was significantly attenuated by TGF-β-induced EMT. The cellular uptake of SN38 was impeded during EMT, which affected the therapeutic efficacy. The combination of LY364947 and cSN38 markedly enhanced the cellular uptake of SN38, increased cytotoxic effects, and inhibited EMT in PDAC cells in vitro. Furthermore, cSN38 + LY significantly inhibited PDAC xenograft growth in vivo. The cSN38 + LY nanoparticles increased the therapeutic efficacy of cSN38 via repressing the EMT of PDAC cells. Our findings provide a rationale for designing nanoscale therapeutics to combat PDAC.

Pore-forming Peptides: A New Treatment Option for Cancer.

Cancer, a challenging medical problem, affects millions of people around the world. Cancer cell resistance is one of the main drawbacks in the complete prosperity of even more sophisticated therapies. Pore-forming peptides (PFPs), a group of natural defense system proteins are used by nearly all living organisms as anti-bacterial and anti-- fungal agents, and could also be regarded as novel tumoricidal peptides. PFPs approach entails using soluble peptides by assembling them mainly on the target cell membrane and forming potential death-causing pores. Physical damage induction by natural PFPs or their synthetic derivatives could conquer the resistance mechanisms of tumor cells. Given that peptide drugs involve a significant proportion of the pharmaceutical market primarily because of easy synthesis and safety, evaluating this nature provided a model system as a group of anticancer peptides seems a valuable approach. Here, the mode of action of PFPs and their anticancer mechanism are highlighted, followed by addressing the anticancer studies using PFPs from different sources along with various strategies applied to obtain selective action of PFPs against cancer cells. Challenges and future perspectives of these promising bioactive molecules in cancer treatment are also provided.

Targeted Alpha Particle Therapy Remodels the Tumor Microenvironment and Improves Efficacy of Immunotherapy

The tumor microenvironment (TME) can severely impair immunotherapy efficacy by repressing the immune system. In a multiple myeloma (MM) murine model, we investigated the impact of targeted alpha particle therapy (TAT) on the immune TME. TAT was combined with an adoptive cell transfer of CD8 T cells (ACT), and the mechanisms of action of this combination were assessed at the tumor site. This combination treatment was conducted in a syngeneic MM murine model grafted subcutaneously. TAT was delivered by intravenous injection of a bismuth-213 radiolabeled anti-CD138 antibody. To strengthen antitumor immune response, TAT was combined with an ACT of tumor-specific CD8+ OT-1 T-cells. The tumors were collected and the immune TME analyzed by flow cytometry, immunohistochemistry, and ex vivo T-cell motility assay on tumor slices. The chemokine and cytokine productions were also assessed by quantitative reverse transcription polymerase chain reaction. Tumor-specific CD8+ OT-1 T cells infiltrated the tumors after ACT. However, only treatment with TAT resulted in regulatory CD4 T-cell drop and transient increased production of interleukin-2, CCL-5, and interferon-γ within the tumor. Moreover, OT-1 T-cell recruitment and motility were increased on tumor slices from TAT-treated mice, as observed via ex vivo time lapse, contributing to a more homogeneous distribution of OT-1 T cells in the tumor. Subsequently, the tumor cells increased PD-L1 expression, antitumor cytokine production decreased, and OT-1 T-cells overexpressed exhaustion markers, suggesting an exhaustion of the immune response. Combining TAT and ACT seems to transiently remodel the cold TME, improving ACT efficiency. The immune response then leads to the establishment of other tumor cell resistance mechanisms.

Systematic Characterization of the “Resistome” of Tumor Cells Against Panels of Small Molecule Inhibitors — Application in the Context of Inhibitors of Transcriptional Cyclin-Dependent Kinases (tCDKs) and Implications for Future “Individualized” Clinical Applications

The development of CRISPR/Cas9-based functional genomic approaches have greatly accelerated the pace of research on characterizing the molecular mechanisms of tumor cell resistance to diverse classes of therapeutics. We reasoned that this technology can be applied to systematically and prospectively identify candidate genes regulating tumor cell sensitivity vs. resistance against a collection of inhibitors with overlapping vs. distinct specificities against different structurally- and functionally-related molecular targets; and that the signature of genes emerging from such genome-wide resistance screens could also exhibit overlapping vs. distinct features consistent with the profile targets of the respective compounds. We applied this hypothesis in the context of a collection of small molecule inhibitors against transcriptional cyclin-dependent kinases (tCDKs). This choice was specifically motivated by our recent observations that a collection of transcription factors selectively drive the biology of multiple myeloma (MM) cells (compared to other neoplasias): while direct and selective small molecule inhibitors against these myeloma-selective transcription factors are not currently available, the expression of some them or their target genes is regulated by tCDKs, such as CDK7. We evaluated the CDK7/12/13 inhibitor THZ-01, the CDK7-selective inhibitor YKL05-124 and the CDK12/13 dual inhibitor MFH2-90-1 (referred herein as YKL and MFH respectively); and confirmed that these inhibitors exhibit in vitro activity against MM cells, with variable potency across cell lines. For our CRISPR/Cas9-based screens, we utilized MM.1S cells engineered to stably express the Cas9 nuclease and lentivirally transduced with the pooled genome-wide sgRNA library Brunello (~70,000 sgRNAs, 4 sgRNAs per gene; plus additional non-targeting control sgRNAs); and exposed the MM.1S-Cas9+ sgRNA+ pools of MM cells to each of these compounds (2 separate screens for THZ-01; 1 for YKL and 1 for MFH). Treatments consistent of successive rounds of treatment (at each compounds IC25 dose for anti-MM activity) for several weeks of culture until treatment-resistant populations of tumor cells emerged. PCR and next-generation sequencing we performed, as in other studies of our group and others, to quantify the relative enrichment or depletion of sgRNAs against different genes in treatment-resistant cells vs. sensitive treatment-naïve cells. We documented concordance of results between the 2 THZ-01 resistance screens; and pronounced overlap, but also distinct differences between the genes associated with resistance or sensitization to THZ vs YKL (consistent with their common effect on CKD7, but the lack of CDK12/13 inhibition by YKL) or between THZ-01 vs. MFH; while more limited overlap was observed between MFH and YKL, consistent with their different specificities. The collection of genes associated with resistance to CDK7 or CDK7/12/13 inhibition contain several recognizable, but previously understudied, members or regulators of the CDK family; as well as a diverse group of other genes with known or putative roles in regulation of cell cycle, cell survival, autophagy, proteostasis and intracellular trafficking. Importantly, both the overlapping and distinct “resistome” signatures identified by CRISPR/Cas9 screens for these compounds were different from “resistome” signatures which we have obtained from similar genomewide CRISPR studies for resistance against other classes of investigational or established therapeutics for MM. Our study exemplifies the feasibility of applying CRISPR/Cas9 gene-editing based treatment resistance screens in order to not only define the molecular networks regulating the sensitivity vs. resistance of tumor cells to the respective treatments, but also to provide insights into known or previously underappreciated aspects of the specificity of these compounds against diverse structurally related targets. We envision that similar applications of this approach can be applied to prospectively identify which molecules mediate the antitumor activity of pharmacological agents for which their targets are not currently defined. DisclosuresMitsiades:Novartis: Research Funding; Abbvie: Research Funding; Ono: Research Funding; TEVA: Research Funding; Janssen/Johnson & Johnson: Research Funding; Takeda: Other: Employment of family member.

The Latest Battles Between EGFR Monoclonal Antibodies and Resistant Tumor Cells.

Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor involved in homeostatic regulation of normal cells and carcinogenesis of epithelial malignancies. With rapid development of the precision medicine era, a series of new therapies targeting EGFR are underway. Four EGFR monoclonal antibody drugs (cetuximab, panitumumab, nimotuzumab, and necitumumab) are already on the market, and a dozen other EGFR monoclonal antibodies are in clinical trials. Here, we comprehensively review the newly identified biological properties and anti-tumor mechanisms of EGFR monoclonal antibodies. We summarize recently completed and ongoing clinical trials of the classic and new EGFR monoclonal antibodies. More importantly, according to our new standard, we re-classify the complex evolving tumor cell resistance mechanisms, including those involving exosomes, non-coding RNA and the tumor microenvironment, against EGFR monoclonal antibodies. Finally, we analyzed the limitations of EGFR monoclonal antibody therapy, and discussed the current strategies overcoming EGFR related drug resistance. This review will help us better understand the latest battles between EGFR monoclonal antibodies and resistant tumor cells, and the future directions to develop anti-tumor EGFR monoclonal antibodies with durable effects.

XCT (SLC7A11) expression confers intrinsic resistance to physical plasma treatment in tumor cells

Cold physical plasma is a partially ionized gas investigated as a new anticancer tool in selectively targeting cancer cells in monotherapy or in combination with therapeutic agents. Here, we investigated the intrinsic resistance mechanisms of tumor cells towards physical plasma treatment. When analyzing the dose-response relationship to cold plasma-derived oxidants in 11 human cancer cell lines, we identified four ‘resistant’ and seven ‘sensitive’ cell lines. We observed stable intracellular glutathione levels following plasma treatment only in the ‘resistant’ cell lines indicative of altered antioxidant mechanisms. Assessment of proteins involved in GSH metabolism revealed cystine-glutamate antiporter xCT (SLC7A11) to be significantly more abundant in the ‘resistant’ cell lines as compared to ‘sensitive’ cell lines. This decisive role of xCT was confirmed by pharmacological and genetic inhibition, followed by cold physical plasma treatment. Finally, microscopy analysis of ex vivo plasma-treated human melanoma punch biopsies suggested a correlation between apoptosis and basal xCT protein abundance. Taken together, our results demonstrate that xCT holds the potential as a biomarker predicting the sensitivity of tumor cells towards plasma treatment.

Peroxynitrite affects MHC I peptide repertoire presented on tumor cells and impedes the efficacy of antitumor cytotoxic T-lymphocytes

Abstract Despite the advancements in enhancing antigen-specific T cell responses in cancer their clinical benefit remains limited. Recent studies demonstrated that tumor microenvironment factors are able to dampen in vivo anti-tumor T cell response. Previously, we found that myeloid cells produce large amounts of peroxynitrite (PNT) in the tumor. The pretreatment with PNT blocked binding of exogeneous peptides by tumor MHC I and decreased tumor killing by cytotoxic T lymphocytes (CTL). We hypothesized that PNT affects tumor MHC I peptide repertoire, resulting in inability of CTL to recognize and eliminate tumor cells. To test this hypothesis, we treated tumor cells with PNT and analyzed MHC I peptides by LC-MS/MS. We found that PNT dramatically affected the repertoire of tumor MHC I peptides. We identified two groups of peptides: “stable” peptides that were retained on MHC class I after PNT treatment and “non-stable” peptides that were significantly reduced after treatment. The characterization of peptides had revealed similar binding capacity for both “stable” and “non-stable” peptides. However, the dissociation rate of “non-stable” peptides from peptide-MHC I complexes was significantly higher. We found that PNT treatment impaired proteasomal activity in tumor cells. This may skew MHC I repertoire towards more “stable” peptides. To examine biological relevance of PNT, we generated CTLs specific to representative “stable” and “non-stable” peptides. We confirmed that PNT pretreatment of tumor cells decreased the killing efficacy of CTLs specific to “non-stable”, but not to “stable” peptides. Overall, our findings demonstrate a novel PNT-mediated mechanism of tumor cell resistance to CTLs that could be targeted for cancer immunotherapy.

Sustained NF-\u03baB-STAT3 signaling promotes resistance to Smac mimetics in Glioma stem-like cells but creates a vulnerability to EZH2 inhibition

Glioblastoma is an incurable and highly aggressive brain tumor. Understanding therapeutic resistance and survival mechanisms driving this tumor type is key to finding effective therapies. Smac mimetics (SM) emerged as attractive cancer therapeutics particularly for tumor populations that are highly resistant to conventional apoptosis-inducing therapies. We evaluated the therapeutic efficacy of SM on Glioma stem-like cells (GSCs) and showed that this family of compounds stimulates an adaptive response triggered by TNFα. Increased expression of TNFα results in a prolonged and sustained activation of NF-κB and STAT3 signaling thus activating several tumor cell resistance mechanisms in GSCs. We show that STAT3 activation is contingent on EZH2 activation and uncover a synergistic lethality between SM and EZH2 inhibitors. Therapeutic inhibition of EZH2 impaired the viability of SM-treated GSCs. Our study outlines the molecular underpinnings of SM resistance in glioblastoma and provides mechanistic insight to overcome this resistance and increase therapeutic efficacy.

Abstract A146: Systematic discovery of immune regulatory mechanisms in tumor cells

Abstract Many human cancers are resistant to immunotherapy for reasons that are poorly understood. We used a genome-scale CRISPR/Cas9 screen to identify mechanisms of tumor cell resistance to killing by cytotoxic T-cells, the central effectors of antitumor immunity. Inactivation of &amp;gt;100 genes sensitized mouse B16F10 melanoma cells to killing by T-cells, including Pbrm1, Arid2 and Brd7, which encode components of the PBAF form of the SWI/SNF chromatin remodeling complex. Loss of PBAF function increased tumor cell sensitivity to interferon-γ, resulting in enhanced secretion of chemokines that recruit effector T-cells. Treatment-resistant tumors became responsive to immunotherapy when Pbrm1 was inactivated. In many human cancers, expression of PBRM1 and ARID2 inversely correlated with expression of T-cell cytotoxicity genes, and Pbrm1-deficient murine melanomas were more strongly infiltrated by cytotoxic T-cells. Citation Format: Deng Pan, Aya Kobayashi, Peng Jiang, Guo-Cheng Yuan, X. Shirley Liu, John Doench, Xintao Qiu, Prakash Rao, Henry Long, Myles A. Brown, Kai W. Wucherpfennig, Lucas Ferrari de Andrade, Rong En Tay, Adrienne M. Luoma, Daphne Tsoucas, Klothilda Lim. Systematic discovery of immune regulatory mechanisms in tumor cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A146.

CRISPR-Based Functional Genomics Studies Reveal Distinct and Overlapping Genes Mediating Resistance to Different Classes of Heterobifunctional Degraders of Oncoproteins: Implications for Novel Therapeutics across Diverse Neoplasias

Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases (e.g. CRBN and VHL) to induce neopmorphic ubiquitination and proteasomal degradation of target oncoproteins, with potent preclinical activity against diverse neoplasias. Despite intense recent efforts to develop pharmacological “degraders” against many different oncoproteins, the mechanisms regulating tumor cell sensitivity to different classes of these “degraders” remain incompletely understood. To address this question in an unbiased manner, we performed genome-scale CRISPR/Cas9-based gene editing loss-of-function (LOF) studies in MM.1S multiple myeloma (MM) cells treated with CRBN-mediated degraders of BET bromodomain proteins (dBET6) or CDK9 (Thal-SNS-032); or with VHL-mediated degraders of BET bromodomain proteins (ARV-771 or MZ-1). We observed that MM cell resistance to any of these “degraders” does not involve genes with recurrent LOF in MM patients and association with high-risk MM (e.g. for TP53, PTEN, negative regulators of cell cycle, et.c.), suggesting that these degraders may exhibit activity against tumor cells with prognostically adverse genetic features.In tumor cells resistant to the CRBN-mediated degraders dBET6 and Thal-SNS-032, we observed significant enrichment of sgRNAs targeting CRBN itself or (to a lesser extent) other components or regulators of its cullin RING ligase (CRLCUL4A) complex, including members of the COP9 signalosome (COPS7A, COPS7B, COPS2, COPS3, COPS8, GPS1, etc.), DDB1, or the E2 ubiquitin conjugating enzyme UBE2G1. In tumor cells resistant to the VHL-mediated degraders MZ-1 and ARV-771, we observed pronounced enrichment of sgRNAs for CUL2, VHL itself, other members (e.g. RBX1, elongin B/C [TCEB1, TCEB2] of the CUL2 complex with VHL), as well as COP9 signalosome genes (COPS7B, COPS8) and UBE2R2. We also validated, using individual sgRNAs for several of these candidate genes that their CRISPR knockout can decrease tumor cell response to dBET6 and Thal-SNS-032 treatment (e.g. for CRBN, COPS7B, COPS2, or COPS8) or MZ-1 and ARV-771 (e.g. for VHL, COP7B and COPS8). Notably, the sgRNAs against COP9 signalosome genes conferred less pronounced decrease in sensitivity to VHL-, than CRBN-based, degraders, suggesting that COP9 signalosome loss has differential roles in the function of CUL4ACRBN vs. CUL2VHL and potentially other CRL complexes. Tumor cells isolated from our CRISPR knockout screens with confirmed resistance to a given degrader were then treated with other degraders operating through the same or different E3 ligase; and against the same or different oncoprotein: we observed cross-resistance between degraders operating through the same E3 ligase against different oncoproteins, but not for degraders targeting the same protein via different E3 ligase/CRLs: this result is consistent with our observation for substantial gene-level differences (despite pathway-level similarities) for resistance mechanisms for CRBN- vs. VHL-based degraders. In conclusion, our study systematically defined at genome-scale the resistance mechanisms of tumor cells against degraders which leverage the same E3 ligase against different targets; or target the same oncoprotein through different E3 ligases/CRL complexes. We observed that for multiple types of degraders, tumor cell resistance is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein. The observed pathway-level similarities and major individual gene-level differences in resistance mechanisms for CRBN- and VHL-mediated degraders likely reflects the different composition and regulation of the respective CRL complexes mediating the action of these classes of degraders Our observations suggest that preventing or delaying resistance to pharmacological degradation of oncoproteins may require concurrent or sequential/alternating use of degraders operating through different E3 ligases and ideally, different CRL complexes; while synthetic lethal strategies to prevent COP9 signalosome LOF may also be contemplated to counteract a common, but quantitatively less pronounced, potential mechanism of resistance for several different classes of degraders. Collectively, our study highlights important new directions in the development of new pharmacological degraders for blood cancers and other neoplasias. DisclosuresRichardson:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Licht:Celgene: Research Funding. Boise:Abbvie: Consultancy; AstraZeneca: Honoraria. Gray:C4 Therapeutics: Consultancy. Mitsiades:TEVA: Research Funding; Janssen/ Johnson & Johnson: Research Funding; EMD Serono: Research Funding; Takeda: Other: employment of a relative; Abbvie: Research Funding.

Abstract B30: Dianhydrogalactitol (VAL-083) has the potential to overcome major challenges in the treatment of DIPG

Abstract Despite several decades of clinical trial, diffuse intrinsic pontine gliomas (DIPG) continue to have a dismal outcome and survival remains dismal. DIPG is inoperable and standard treatment is radiation alone, as the addition of chemotherapy has not improved survival. Major obstacles to the successful treatment of DIPG include an intact blood-brain barrier impeding drug penetration and inherent tumor-cell resistance mechanisms to chemotherapeutics. Dianhydrogalactitol (VAL-083) is a structurally unique bifunctional DNA targeting agent that readily crosses the blood-brain barrier. VAL-083 forms interstrand DNA crosslinks at the N7 position of guanine, leading to persistent and irreversible DNA double-strand breaks, cell cycle arrest, and ultimately cancer cell death. VAL-083 has cytotoxic activity in several pediatric brain tumors as assessed in historical NCI-sponsored clinical trials, both as a single agent and in combination with other chemotherapeutics. We have previously demonstrated that VAL-083 is able to overcome chemoresistance mediated by DNA repair protein O6-methylguanine DNA methyltransferase (MGMT). Expression of MGMT is strongly correlated with resistance to temozolomide (TMZ), which is commonly used in combination with radiation for the treatment of adult brain tumors. VAL-083 activity is also independent of DNA mismatch repair (MMR) system in vitro, a secondary mechanism of resistance to TMZ. VAL-083 potentiates the effect of radiation in TMZ-resistant adult glioblastoma (GBM) cells in vitro and overcomes TMZ resistance in GBM cancer stem cells (CSCs) and non-CSCs. Additionally, VAL-083 demonstrated synergistic efficacy with inhibitors of topoisomerase 1 (camptothecin) and topoisomerase 2 (etoposide) against multiple cancer cell lines. VAL-083’s ability to cross the blood-brain barrier and its ability to overcome common resistance mechanisms, combined with its radiotherapy-potentiating effects, suggest that VAL-083 may provide a new treatment option for DIPG and other pediatric CNS tumors as a single agent, in combination with radiotherapy, or as part of a combination regimen with topoisomerase inhibitors. We recently completed a phase I/II clinical trial in refractory GBM and established a well-tolerated dosing regimen of VAL-083 in adult brain tumor patients. In the present study, we investigated the effects of VAL-083 in combination with radiation or irinotecan (topoisomerase 1 inhibitor) in a panel of DIPG cell lines as well as patient-derived xenografts models. The results will guide a potential clinical trial of VAL-083 in treatment of DIPG, either as part of a chemo-radiation regimen or in combination with topoisomerase inhibitors. Citation Format: Anne Steino, Beibei Zhai, Beibei Zhai, Jeffrey Bacha, Dennis Brown, Shaun Fouse, Joe Costello, Mads Daugaard, Mads Daugaard, Sabine Mueller. Dianhydrogalactitol (VAL-083) has the potential to overcome major challenges in the treatment of DIPG [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B30.

MicroRNAs in cancer drug resistance: Basic evidence and clinical applications.

Development of drug resistance has considerably limited the efficacy of cancer treatments, including chemotherapy and targeted therapies. Hence, understanding the molecular mechanisms underpinning the innate or the acquired resistance to these therapies is critical to improve drug efficiency and clinical outcomes. Several studies have implicated microRNAs (miRNA) in this process. MiRNAs repress gene expression by specific binding to complementary sequences in the 3' region of target messenger RNAs (mRNAs), followed by target mRNA degradation or blocked translation. By targeting molecules specific to a particular pathway within tumor cells, the new generation of cancer treatment strategies has shown significant advantages over conventional chemotherapy. However, the long-term efficacy of targeted therapies often remains poor, because tumor cells develop resistance to such therapeutics. Targeted therapies often involve monoclonal antibodies (mAbs), such as those blocking the ErB/HER tyrosine kinases, epidermal growth factor receptor (cetuximab) and HER2 (trastuzumab), and those inhibiting vascular endothelial growth factor receptor signaling (e.g., bevacizumab). Even though these are among the most used agents in tumor medicine, clinical response to these drugs is reduced due to the emergence of drug resistance as a result of toxic effects in the tumor microenvironment. Research on different types of human cancers has revealed that aberrant expression of miRNAs promotes resistance to the aforementioned drugs. In this study, we review the mechanisms of tumor cell resistance to mAb therapies and the role of miRNAs therein. Emerging treatment strategies combine therapies using innovative miRNA mimics or antagonizers with conventional approaches to maximize outcomes of patients with cancer.

Abstract 1008: Peroxynitrite mediates the resistance of tumor cells to cytotoxic T cells by altering the MHC1-peptide repertoire on tumor cells

Abstract Therapeutic advances were able to significantly enhance antigen-specific T cell responses in cancer therapy. However, despite the obvious success, our ability to translate the generation of specific T-cell response into a clinical benefit remains limited. Recently, factors of tumor microenvironment such as hypoxia and oxidative agents were shown to dampen the efficiency of T-cell responses. Previously, we demonstrated that macrophages and myeloid-derived suppressor cells (MDSCs) produce a significant amount of highly oxidative agent, peroxynitrite (PNT), inside the tumor and revealed that PNT inhibited binding of processed peptides to tumor cell MHC class I (MHC I). As a result, tumor cells become resistant to antigen-specific cytotoxic T cells (CTLs). In other studies, it was shown that high levels of PNT (measured by nitrotyrosine level) in tumor tissues are associated with worse clinical outcome. We hypothesized that PNT affects peptide repertoire presented by tumor cell MHC I molecules and by this impairs the immune recognition of tumor cells by CTLs. To test this hypothesis we compared MHC I peptides isolated from PNT-treated and nontreated tumor cells. Quantitative LC-MS/MS analysis revealed that PNT treatment affected the peptide repertoire of MHC I bound peptides. A number of peptides were substantially underrepresented in PNT-treated cells (“nonstable” peptide subset), whereas others remained at the same level after PNT treatment (“stable” peptide subset). We generated CTLs specific to some peptides from “stable” or “nonstable” groups. PNT treatment significantly decreased the cytotoxic activity of T cells specific to “non-stable” but not to “stable” peptides, confirming our initial hypothesis. To further characterize “stable” and “nonstable” peptides we investigated their affinity to MHC I as well as the stability of the formed peptide-MHC I (pMHC) complexes. We found that peptides from both groups demonstrated equally high affinity to MHC I. However, we observed significantly higher dissociation rate of pMHC complexes formed by peptides from “nonstable” group than by “stable” peptides. PNT treatment disrupted proteasome function in tumor cells, suggesting that PNT may impair antigen-processing machinery and “nonstable” peptides quickly dissociating from MHC I were not recovered by malfunctioning proteasome. Consecutively, this could lead to the poorer peptide presentation on the tumor cell surface hindering tumor cell recognition and killing by CTLs. Our findings demonstrate a novel PNT-mediated mechanism of tumor cell resistance to specific CTLs and suggest potential therapeutic avenues for its neutralization. Citation Format: Evgenii N. Tcyganov, Dmitry I. Gabrilovich. Peroxynitrite mediates the resistance of tumor cells to cytotoxic T cells by altering the MHC1-peptide repertoire on tumor cells [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 1008.

Abstract 900: Dianhydrogalactitol (VAL-083) has the potential to overcome major challenges in the treatment of DIPG

Abstract Despite decades of clinical trials, children with diffuse intrinsic pontine gliomas (DIPG) continue to have a very poor prognosis and dismal survival. DIPG is inoperable and standard treatment is radiation alone. Major obstacles to the successful treatment of DIPG include 1) an intact blood brain barrier impeding drug penetration, 2) inherent tumor cell resistance mechanisms to conventional chemotherapeutics, and 3) a lack of drug-induced potentiation of radiotherapy. VAL-083 is a structurally unique bi-functional DNA targeting agent that has the potential to overcome these barriers. VAL-083 readily crosses the blood-brain barrier and accumulates in brain tumor tissue where it rapidly forms interstrand DNA crosslinks at guanine-N7, leading to persistent DNA double strand breaks, cell cycle arrest, and cancer cell death. VAL-083 has cytotoxic activity in several pediatric brain tumors as assessed in NCI-sponsored clinical trials, both as a single agent and in combination with other chemotherapeutics. We recently completed a Phase I/II clinical trial in refractory GBM and established a well-tolerated dosing regimen of VAL-083 in adult brain tumor patients. We have previously demonstrated that VAL-083 cytotoxicity is independent of both the primary, DNA repair protein O6-methylguanine DNA methyltransferase (MGMT), and the secondary, DNA mismatch repair (MMR) system, mechanisms of resistance to temozolomide (TMZ) in vitro. TMZ is commonly used in combination with radiation for the treatment of adult brain tumors but has failed to improve the effect of radiation in DIPG. Importantly, VAL-083 potentiates the effect of radiation and overcomes TMZ resistance in TMZ-resistant adult glioblastoma cancer stem cells (CSCs) and non-CSCs at concentrations that are physiologically achievable in the CNS. Additionally, VAL-083 demonstrated synergistic efficacy in prostate and lung cancer cells with topoisomerase (Topo) inhibitors camptothecin and etoposide, commonly used in the treatment of pediatric brain tumors. In conclusion, VAL-083 is able to 1) cross the blood brain barrier, 2) overcome resistance mechanisms to chemotherapeutics commonly used in the treatment of adult gliomas, and 3) potentiate the effects of radiation in adult glioma cells at physiologically achievable concentrations. Thus, VAL-083 may have the potential to overcome major challenges in DIPG treatment as a single agent, in combination with radiotherapy, or as part of a combination regimen with Topo inhibitors. In the present study, we investigated the effects of VAL-083 in combination with radiation, irinotecan (Topo 1 inhibitor), or Wee1 inhibitor AZD1775 in a panel of DIPG cell lines with varying genetic profiles. The results will provide guidance for the design of animal models studying VAL-083 treatment of DIPG, either as part of a chemo-radiation regimen or in combination with Topo or Wee1 inhibitors. Citation Format: Anne Steino, Beibei Zhai, Jeffrey A. Bacha, Dennis M. Brown, Mads Daugaard, Sabine Mueller. Dianhydrogalactitol (VAL-083) has the potential to overcome major challenges in the treatment of DIPG [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 900.

Redirecting T cells to eradicate B-cell acute lymphoblastic leukemia: bispecific T-cell engagers and chimeric antigen receptors

Recent advances in antibody technology to harness T cells for cancer immunotherapy, particularly in the difficult-to-treat setting of relapsed/refractory acute lymphoblastic leukemia (r/r ALL), have led to innovative methods for directing cytotoxic T cells to specific surface antigens on cancer cells. One approach involves administration of soluble bispecific (or dual-affinity) antibody-based constructs that temporarily bridge T cells and cancer cells. Another approach infuses ex vivo-engineered T cells that express a surface plasma membrane-inserted antibody construct called a chimeric antigen receptor (CAR). Both bispecific antibodies and CARs circumvent natural target cell recognition by creating a physical connection between cytotoxic T cells and target cancer cells to activate a cytolysis signaling pathway; this connection allows essentially all cytotoxic T cells in a patient to be engaged because typical tumor cell resistance mechanisms (such as T-cell receptor specificity, antigen processing and presentation, and major histocompatibility complex context) are bypassed. Both the bispecific T-cell engager (BiTE) antibody construct blinatumomab and CD19-CARs are immunotherapies that have yielded encouraging remission rates in CD19-positive r/r ALL, suggesting that they might serve as definitive treatments or bridging therapies to allogeneic hematopoietic cell transplantation. With the introduction of these immunotherapies, new challenges arise related to unique toxicities and distinctive pathways of resistance. An increasing body of knowledge is being accumulated on how to predict, prevent, and manage such toxicities, which will help to better stratify patient risk and tailor treatments to minimize severe adverse events. A deeper understanding of the precise mechanisms of action and immune resistance, interaction with other novel agents in potential combinations, and optimization in the manufacturing process will help to advance immunotherapy outcomes in the r/r ALL setting.

DeBouganin Diabody Fusion Protein Overcomes Drug Resistance to ADCs Comprised of Anti-Microtubule Agents.

Antibody drug conjugates (ADC), comprised of highly potent small molecule payloads chemically conjugated to a full-length antibody, represent a growing class of therapeutic agents. The targeting of cytotoxic payloads via the specificity and selectivity of the antibody has led to substantial clinical benefits. However, ADC potency can be altered by mechanisms of resistance such as overexpression of efflux pumps or anti-apoptotic proteins. DeBouganin is a de-immunized variant of bouganin, a ribosome-inactivating protein (RIP) that blocks protein synthesis, thereby leading to apoptosis. When conjugated to trastuzumab (T-deB), deBouganin was more potent than ado-trastuzumab-emtansine (T-DM1) and unaffected by resistance mechanisms to which DM1 is susceptible. To further highlight the differentiating mechanism of action of deBouganin, HCC1419 and BT-474 tumor cells that survived T-DM1 or trastuzumab-MMAE (T-MMAE) treatment were treated with an anti-HER2 C6.5 diabody–deBouganin fusion protein or T-deB. C6.5 diabody–deBouganin and T-deB were potent against HCC1419 and BT-474 cells that were resistant to T-DM1 or T-MMAE killing. The resistant phenotype involved MDR pumps, Bcl-2 family members, and the presence of additional unknown pathways. Overall, the data suggest that deBouganin is effective against tumor cell resistance mechanisms selected in response to ADCs composed of anti-microtubule payloads.