Mechanisms Of Therapy Resistance Research Articles

Abstract 4200: A novel 3D culture platform, E-slice, retains intact tumor microenvironment and tumor infiltrating immune cells

Abstract Human cancer tissue is a complex ecosystem with heterogeneous cell types that communicate with each other dynamically to promote tumor progression and immune evasion. Hence, experimental systems that destroy or select for cancer cells in culture have often failed to accurately predict clinical responses of new drugs. Therefore, a technology that can retain the intact tumor microenvironment (TME) and accurately predict cancer patient’s response to anti-cancer treatments, including immunotherapies, will greatly benefit patients and dramatically reduce the cost of new drug development. E-slice is a proprietary 3D human tumor culture platform that maintains individual patient tumor’s unique ecosystem and the TME ex vivo for up to 30 days. It is generated by making thin sections of intact, fresh tumor tissues and culturing them in serum-free, defined media. As such, immune components and the TME in E-slices faithfully recapitulate human tumors ex vivo. We demonstrate that E-slices can be used to: 1) measure viability changes upon anti-cancer agent treatment longitudinally; 2) retain the native TME and tissue architecture since E-slices are never dissociated or artificially reconstituted; 3) culture any solid tumor types from patient needle biopsy cores or surgical samples, PDX, or mouse models; 4) perform high content drug screening on human tissues; 5) discover secreted biomarkers and to 6) discover or validate molecular mechanisms of action or therapy resistance; and 7) measure immunotherapy responses ex vivo from human tissues. Importantly, it has been shown to accurately predict individual patient treatment responses to chemotherapies and targeted therapies in 4-8 days, paving the way for evidence-based personalized treatment selections in a clinically actionable time frame. Furthermore, single cell RNA-sequencing analysis demonstrates that E-slices can retain the viability and molecular phenotypes of tumor infiltrating immune cells for up to 8 days ex vivo, providing a unique platform to study human tumor-immune interactions and evaluate efficacy of immune modulators on human tumor tissues ex vivo. Citation Format: Viridiana Leyva-Aranda, Thomas Gallup, Jose Maldonado, Corina Margain, Sang Yun, David Gallup, Min Kim, Kyuson Yun. A novel 3D culture platform, E-slice, retains intact tumor microenvironment and tumor infiltrating immune cells [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 4200.

Abstract 7557: Hypoxia-induced downregulation of MUC16: A key player in bispecific T-cell engager resistance in ovarian cancer

Abstract Background: Ovarian cancer (OC) is a lethal disease due to limited treatment options and the development of multidrug resistance. Bispecific T-cell engager (BiTE) therapy represents a promising immunotherapeutic approach to OC. Our previous findings suggest that resistance to BiTE therapy in OC is a complex interplay of factors including loss of tumor-associated antigens and epithelial-mesenchymal transition (EMT). Since the peritoneum is a relatively hypoxic (HX) tumor microenvironment, we hypothesized that HX plays a role in the modulation of MUC16 and potential resistance to BiTE therapy. Methods: After informed consent, samples were collected from patients with recurrent OC undergoing treatment with MUC16 BiTEs. For HX studies, we used OVCAR3 tumor cell lines which express full-length MUC16. Tumor cells were cultured under normoxic (NX) or HX conditions. The effect of HX on cell death was analyzed using an apoptosis assay. Cells were evaluated for changes in cell surface MUC16 expression by flow cytometry. MUC16 protein expression and EMT markers were analyzed by western blot. Proteasome inhibitors were used to stabilize MUC16 expression under HX conditions. Antibody internalization assays were conducted to assess MUC16 endocytosis and recycling. Results: In patients who progressed on MUC16 BiTEs, we found decreased MUC16 expression on tumor samples isolated from ascites samples. Using OVCAR3 cells, we found significantly decreased MUC16 expression on ovarian cancer cells grown under HX conditions (Fold change: 0.4 vs NX, p<0.001). Flow cytometry analysis revealed a time-dependent loss of MUC16 expression on the cell surface under HX (64.22% after 9 days HX vs 88.26% NX), highlighting the susceptibility of MUC16 to HX-induced downregulation. Treatment with proteasome inhibitors partially rescued MUC16 expression under HX conditions (Fold change: 1.4 vs NX, p<0.001). Notably, HX did not induce necrotic cell death in these tumor cells. In addition to the loss of MUC16, tumor cells under HX conditions showed alterations in EMT markers. E-cadherin was downregulated with prolonged HX exposure (Fold change: 0.3 vs NX, p < 0.001), while Vimentin (Fold change: 7.1 vs NX, p < 0.001) and Snail (Fold change: 2.8 vs NX, p < 0.001) were upregulated, indicating a potential shift towards a more aggressive phenotype. Epidermal growth factor receptor (EGFR) was also upregulated (Fold change: 4.9 vs NX, p < 0.001), potentially contributing to enhanced survival and invasion of OC cells under HX stress. Finally, under HX conditions, MUC16 internalization was slower in response to antibody binding over time due to reduced cell antigen expression, corroborating the observed MUC16 loss. Conclusion: Understanding hypoxia-induced MUC16 downregulation and its downstream effects on EMT markers and CA125 expression provides crucial insight into the mechanisms of BiTE therapy resistance in ovarian cancer. Citation Format: Mengyao Xu, Syem K. Barakzai, Artem Kononenko, Raj Kumar, Irva Veillard, Eugene Kim, Timothy S. Bond, David R. Spriggs, Bo R. Rueda, Oladapo Yeku. Hypoxia-induced downregulation of MUC16: A key player in bispecific T-cell engager resistance in ovarian cancer [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 7557.

Abstract 6459: Molecular profiling of advanced non-small cell lung cancer in response to first-line immune checkpoint inhibitors and/or chemotherapy using multimodal real-world data

Abstract Background: Up to 85% of lung cancer patients have non-small cell lung cancer (NSCLC). Standard of care for NSCLC includes immune checkpoint inhibitors (ICI) and/or platinum-based chemotherapy (CT). However, therapy success is limited with up to 70% of patients not responding to ICI. Mechanisms of resistance across treatments are not fully understood. In this study, we explore real-world data to characterize molecular and genomic changes in advanced NSCLC treated with ICI, ICI+CT, or CT. Methods: Stage III/IV NSCLC patients receiving first-line (1L) ICI, ICI+CT, or CT were identified from Tempus Labs, Inc.’s de-identified, multimodal real-world database. Patients’ solid tissue biopsies taken pre- or post-1L underwent next generation sequencing with the Tempus xT assays (DNAseq of 648 genes at 500x coverage and whole transcriptome RNAseq). Patients’ clinical and outcome data were abstracted from electronic health records. We analyzed changes in gene expression, pathway enrichment, estimated immune cell components, and somatic mutations in responders (complete/partial response) and non-responders (progression/stable disease) upon 1L treatment. Results: 1L non-responders (70%, 51%, and 47%) were identified across cohorts (143 ICI, 764 ICI+CT, and 228 CT, respectively). 58% of pre-1L biopsies were taken from distal metastatic tissues. Distinct pathways altered post-1L include the downregulated E2F target pathway in CT cohort, and upregulated TNFα/NFκB signaling and the hypoxia and protein secretion pathways in ICI cohort. Significant (FDR < 0.05) augmented expression was observed for 90, 23, and 65 genes in non-responders vs. responders post ICI, ICI+CT, and CT, respectively. RNAseq deconvolution reveals decreased plasma and CD4+ activated T cell fraction in metastatic biopsies, whereas increased CD4+ activated T cell and monocyte fraction was seen after treatment only in ICI cohort. Genes with oncogenic driver mutations reported in NSCLC were detected including TP53 (68%), KRAS (30%), STK11 (13%), EGFR (11%), CDKN2A (10%), KEAP1 (9%), and NF1 (7%). Of the 250 mutated genes, 72 (29%) were commonly not detected post-1L, whereas 38, 3, and 8 unique genes with loss of detectable mutations were identified post-1L of ICI, ICI+CT, and CT, respectively. Since loss of detectable mutations is a sign of loss of subpopulations of cancer cells hosting such mutations that succumb to therapy, these genetic mutations could be used to select patients who can benefit from such therapy. Conclusions: Distinct molecular characteristics including tumor immune components and genomic alterations are associated with 1L response in advanced NSCLC. Real-world data analyses provide opportunity to explore distinct mechanisms of therapy resistance and identify potential biomarkers of therapy response for further validation in clinical studies. Citation Format: Xiaoyong Fu, Jordan Kardos, Paola Correa, Min Wang, Li, Jackson Egen, Shahed Iqbal. Molecular profiling of advanced non-small cell lung cancer in response to first-line immune checkpoint inhibitors and/or chemotherapy using multimodal real-world data [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 6459.

Abstract 1678: Docetaxel induces Rac1 and Cdc42 Rho GTPase activation in prostate cancer cells

Abstract Prostate cancer (PCa) is a major health burden that affects the quality of life of men worldwide. In the United States, PCa is the second leading cause of mortality among cancers that affect men. Treatment of advanced PCa aims to suppress androgen signaling and proliferation in cancer cells using anti-androgens and chemotherapy. However, the development of drug resistance is a major limitation to curing advanced prostate cancer and strategies to block mechanisms of therapy resistance would enhance treatment efficacy and potentially improve patient prognosis. Cdc42 and Rac1, members of the family of Rho GTPases, have been associated with the progression of different types of hormone-associated cancer, such as breast, ovary, and prostate, among others. The current study was designed to evaluate the role of Cdc42 and Rac1 in chemoresistant prostate cancer cells, including under castration-resistant and androgen-independent conditions. We did not observe significant differences in active (Rac-GTP) or total Rac1 levels in chemoresistant compared to parental PC3 or DU145 cells; similar findings were observed for Ccd42. Treatment of drug-naïve cells with a sublethal dose of docetaxel (10uM) induced gradual increases in Rac1-GTP up to 30 minutes in 22rv1, 60 minutes in LNCaP and DU145, and 24 hours in PC3 cells. Cdc42-GTP levels also increased with subacute docetaxel treatment up to 24 hours in 22rv1 and DU145. Chemoresistant 22rv1, PC3, and DU145 exhibited similar sensitivity to Rac1 and Ccd42 inhibitors including MB1-167, MBQ-168, EHop-097, with an IC50 of about 1uM for each compound and line. Ongoing studies are evaluating the role of these Rho-GTPases in 3-dimensional growth and survival. Citation Format: Andrea Paola López González, Jessica Colón González, María C. Santa María Fuentes, Surangani Dharmawardhane, Carlos J. Diaz Osterman. Docetaxel induces Rac1 and Cdc42 Rho GTPase activation in prostate cancer cells [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 1678.

P53 modulates kinase inhibitor resistance and lineage plasticity in NF1-related MPNSTs.

Malignant peripheral nerve sheath tumors (MPNSTs) are chemotherapy resistant sarcomas that are a leading cause of death in neurofibromatosis type 1 (NF1). Although NF1-related MPNSTs derive from neural crest cell origin, they also exhibit intratumoral heterogeneity. TP53 mutations are associated with significantly decreased survival in MPNSTs, however the mechanisms underlying TP53-mediated therapy responses are unclear in the context of NF1-deficiency. We evaluated the role of two commonly altered genes, MET and TP53, in kinome reprograming and cellular differentiation in preclinical MPNST mouse models. We previously showed that MET amplification occurs early in human MPNST progression and that Trp53 loss abrogated MET-addiction resulting in MET inhibitor resistance. Here we demonstrate a novel mechanism of therapy resistance whereby p53 alters MET stability, localization, and downstream signaling leading to kinome reprogramming and lineage plasticity. Trp53 loss also resulted in a shift from RAS/ERK to AKT signaling and enhanced sensitivity to MEK and mTOR inhibition. In response to MET, MEK and mTOR inhibition, we observed broad and heterogeneous activation of key differentiation genes in Trp53-deficient lines suggesting Trp53 loss also impacts lineage plasticity in MPNSTs. These results demonstrate the mechanisms by which p53 loss alters MET dependency and therapy resistance in MPNSTS through kinome reprogramming and phenotypic flexibility.

Biological insights from plasma proteomics of non-small cell lung cancer patients treated with immunotherapy.

Immune checkpoint inhibitors have made a paradigm shift in the treatment of non-small cell lung cancer (NSCLC). However, clinical response varies widely and robust predictive biomarkers for patient stratification are lacking. Here, we characterize early on-treatment proteomic changes in blood plasma to gain a better understanding of treatment response and resistance. Pre-treatment (T0) and on-treatment (T1) plasma samples were collected from 225 NSCLC patients receiving PD-1/PD-L1 inhibitor-based regimens. Plasma was profiled using aptamer-based technology to quantify approximately 7000 plasma proteins per sample. Proteins displaying significant fold changes (T1:T0) were analyzed further to identify associations with clinical outcomes using clinical benefit and overall survival as endpoints. Bioinformatic analyses of upregulated proteins were performed to determine potential cell origins and enriched biological processes. The levels of 142 proteins were significantly increased in the plasma of NSCLC patients following ICI-based treatments. Soluble PD-1 exhibited the highest increase, with a positive correlation to tumor PD-L1 status, and, in the ICI monotherapy dataset, an association with improved overall survival. Bioinformatic analysis of the ICI monotherapy dataset revealed a set of 30 upregulated proteins that formed a single, highly interconnected network, including CD8A connected to ten other proteins, suggestive of T cell activation during ICI treatment. Notably, the T cell-related network was detected regardless of clinical benefit. Lastly, circulating proteins of alveolar origin were identified as potential biomarkers of limited clinical benefit, possibly due to a link with cellular stress and lung damage. Our study provides insights into the biological processes activated during ICI-based therapy, highlighting the potential of plasma proteomics to identify mechanisms of therapy resistance and biomarkers for outcome.

Integrated Proteogenomics Uncover Mechanisms of Glioblastoma Evolution, Pointing to Novel Therapeutic Targets.

Nearly all glioblastoma (GBM) patients relapse following standard treatment and eventually succumb to disease. While large-scale, integrated multiomic studies have tremendously advanced the understanding of primary GBM at the cellular and molecular level, the posttherapeutic trajectory and biological properties of recurrent GBM remain poorly understood. This knowledge gap was addressed in a recent Cancer Cell article in which Kim and colleagues report on a highly integrative proteogenomic analysis performed on 123 matched primary and recurrent GBMs that uncovered a dramatic evolutionary shift from a proliferative state at initial diagnosis to the activation of neuronal and synaptogenic pathways at recurrence following therapy. Neuronal transition was characterized by posttranslational activation of WNT/PCP signaling and BRAF kinase, while many canonical oncogenic pathways, and EGFR in particular, were downregulated. Parallel multiomics analyses of patient-derived xenograft (PDX) models corroborated this evolutionary trajectory, allowing in vivo experiments for translational significance. Notably, targeting BRAF kinase disrupted both the neuronal transition and migration capabilities of recurrent gliomas, which were key characteristics of posttreatment progression. Furthermore, combining BRAF inhibitor vemurafenib with temozolomide prolonged survival in PDX models. Overall, the results reveal novel biological mechanisms of GBM evolution and therapy resistance, and suggest promising therapeutic intervention.

Abstract A007: Representative sampling of invasive breast carcinomas: Interim report from a prospective study (HOLST-F)

Abstract Background: Although a range of therapeutic options are available in the management of invasive breast carcinomas, spatial segregation of tumour subclones has hampered biomarker identification in single-region samples. Representative sampling (RS) overcomes spatial bias by sampling from a homogenized and well-mixed cancer specimen. The Homogenization of Leftover Surgical Tissue Feasibility Study (HoLST-F - NCT03832062) is a prospective trial aiming to assess the feasibility of RS in tumor tissue leftover after pathology sampling. An interim analysis of the breast cancer cohort is presented. Methods: In the context of the HoLST-F study and pre-specified end-points, representative samples derived from 75 leftover invasive breast carcinoma specimens underwent flow cytometric analysis of CK8/18, CD3, Ki67 and DAPI. Tumor cell enrichment by CK8/18 positivity and ploidy (in aneuploid carcinomas) was performed for downstream DNA extraction and whole exome sequencing. Somatic variants were determined using a bespoke pipeline to remove artefacts associated with fixation. Variant oncogenicity and therapeutic evidence levels were assigned by OncoKB variant annotation. Quantitative image analysis was applied to tissue sections from diagnostic FFPE blocks stained with Ki67 immunohistochemistry (n=78) and H&E for lymphocytic infiltration scores (n=175) to correlate with flow cytometry. Results: In enriched representative samples, oncogenic mutations were commonly identified at high variant allele frequency (VAF) in known breast carcinoma driver genes including PIK3CA, CDH1, TP53, KMT2C and GATA3. Other clinically relevant oncogenic mutations were identified in ESR1, PTEN, ERBB2, RB1, AKT1, BRCA1, NF1 and FOXA1 which have been associated with resistance to various anti-cancer therapies. These mutations occurred at low and high VAFs indicative of both of clonal and sub-clonal resistance mechanisms. Recurrent variants in ESR1 (e.g. D538G) and PIK3CA (e.g. H1047R/L) were associated with Level 1 evidence for use as predictive biomarkers, while variants in twelve other genes across the cohort were associated with Level 2-4 evidence. Lymphocyte infiltration scores and Ki67 expression varied by tumor region, however RS by flow cytometry showed strong correlation with a weighted average across multiregional quantification of Ki67 expression (R=0.81, p=2.8 × 10-8) and lymphocyte infiltration (R=0.61, p<2.2 × 10-16). Conclusions: RS of invasive breast carcinoma in the HoLST-F trial has recapitulated the expected genomic driver landscape in breast cancer, in addition to identifying both clonal and subclonal genomic mechanisms of therapy resistance. Many of these mutations are either current or emerging therapeutic targets. Flow quantification of cells expressing phenotypic biomarkers (Ki67 and CD3) is feasible through RS and early analysis indicates that RS correlates with a weighted average across multiple regions. Leftover surgical tissue is an underutilized resource for biomarker assessment in breast carcinomas and can be examined by RS. Citation Format: Brian Hanley, Lisa Gallegos, Lavinia Spain, Husayn Pallikonda, Zayd Tippu, Samantha Hill, Aoune Barhoumi, Fiona Byrne, Yulia Dogva, Ashley Gilchrist, Glenn Noel-Storr, Hannah Veloz, Stacey Stanislaw, Harold Sansano, Kim Edmonds, Eleanor Carlyle, Nicholas Turner, James Larkin, Nelson Alexander, Samra Turajlic. Representative sampling of invasive breast carcinomas: Interim report from a prospective study (HOLST-F) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A007.

Abstract A063: CancerTools.org: Research Tools Supporting Breast Cancer Studies

Abstract CancerTools.org is the latest, non-profit cancer-focused biorepository. Driven by a mission to accelerate cancer research discoveries, the platform allows scientists to deposit and access tangible laboratory research materials. Till date, researchers from over 175 academic institutes globally have already deposited their tools, generating a cutting-edge portfolio of over 5,000 reagents, including a range of cell-based models and antibodies. Breast cancer is one of the most frequent malignancies in women worldwide. A comprehensive collection of tools has been curated at CancerTools.org to support this important area of research. Cell-based models include the RMAM007-3 breast cancer organoid which recapitulates in vivo tumour characteristics and is suitable for disease modelling, 3D imaging, and anticancer drug discovery. Cell lines such as human anti-oestrogen resistant lines developed from MCF-7 and T47D cells, demonstrate resistance to hormone-dependent treatments and are useful for studying therapy resistance mechanisms and biomarker development for breast cancer detection. NCI-HPN-F murine cell lines with recurrent chromosomal aneuploidies, have enabled investigation and identification of tumorigenesis related cancer-specific genes and signalling pathways. Further models include gene-edited cell lines to explore BRCA1 hypermethylation pathways; reporter breast cancer cell lines to assess apoptosis, necrosis, and autophagy; and immortalised human breast epithelial cell lines to study differentiation and phenotypic transformation. To provide a physiologically relevant cell culture environment, PlasmaxTM, a defined media that mirrors the concentration of over 50 components to human plasma, is also part of the repertoire. It has been used to culture triple-negative breast cancer cell lines to recapitulate the tumour metabolic environment. Antibodies to study breast cancer biology, including tumour suppressors, angiogenesis and metastasis, form part of the portfolio. Antibodies targeting tumour suppressors include RB1, which is often lost by mutation, deletion, transcriptional silencing and hyper-phosphorylation in malignancies- including breast cancer. Deletion and rearrangement of RB1 is estimated in ~20-25% of breast cancer cell lines. Tools identifying specific RB1 phosphorylation sites, include Anti-Phospho-Rb for Ser608 and Thr356. Additional insights into breast cancer prognosis are offered by Anti-Maspin [16F7] which recognises wildtype and denaturated forms of the protein. Maspin is an inhibitor of serine proteases with tumour suppressor activity in breast cancer. Its nuclear staining associates with favourable outcomes in contrast to cytoplasmic staining. MUC1 is also aberrantly overexpressed in 90% of human breast cancer, thus, representing a promising marker for breast cancer diagnosis and prognosis. Further research support is offered by an extensive range of antibodies for MUC1’s different isoforms. Visit our stand to explore more about depositing and accessing tools and technologies that support breast cancer research. Citation Format: Vera Moiseeva, Sara Omenetti, Abby Henney, Katie Valencia. CancerTools.org: Research Tools Supporting Breast Cancer Studies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A063.

Oral SERD, a Novel Endocrine Therapy for Estrogen Receptor-Positive Breast Cancer.

Breast cancer is the most common cancer among women worldwide, and estrogen receptor-positive (ER+) breast cancer accounts for a significant proportion of cases. While various treatments are available, endocrine therapies are often the first-line treatment for this type of breast cancer. However, the development of drug resistance poses a significant challenge in managing this disease. ESR1 mutations have been identified as a common mechanism of endocrine therapy resistance in ER+ breast cancer. The first-generation selective estrogen receptor degrader (SERD) fulvestrant has shown some activity against ESR1 mutant tumors. However, due to its poor bioavailability and need for intramuscular injection, it may not be the optimal therapy for patients. Second-generation SERDs were developed to overcome these limitations. These newer drugs have improved oral bioavailability and pharmacokinetics, making them more convenient and effective for patients. Several oral SERDs are now in phase III trials for early and advanced ER+ breast cancer. This review summarizes the background of oral SERD development, the current status, and future perspectives.

Mechanisms of Anti-PD Therapy Resistance in Digestive System Neoplasms.

Digestive system neoplasms are highly heterogeneous and exhibit complex resistance mechanisms that render anti-programmed cell death protein (PD) therapies poorly effective. The tumor microenvironment (TME) plays a pivotal role in tumor development, apart from supplying energy for tumor proliferation and impeding the body's anti-tumor immune response, the TME actively facilitates tumor progression and immune escape via diverse pathways, which include the modulation of heritable gene expression alterations and the intricate interplay with the gut microbiota. In this review, we aim to elucidate the mechanisms underlying drug resistance in digestive tumors, focusing on immune-mediated resistance, microbial crosstalk, metabolism, and epigenetics. We will highlight the unique characteristics of each digestive tumor and emphasize the significance of the tumor immune microenvironment (TIME). Furthermore, we will discuss the current therapeutic strategies that hold promise for combination with cancer immune normalization therapies. This review aims to provide a thorough understanding of the resistance mechanisms in digestive tumors and offer insights into potential therapeutic interventions.

RAD54L2 counters TOP2-DNA adducts to promote genome stability.

The catalytic cycle of topoisomerase 2 (TOP2) enzymes proceeds via a transient DNA double-strand break (DSB) intermediate termed the TOP2 cleavage complex (TOP2cc), in which the TOP2 protein is covalently bound to DNA. Anticancer agents such as etoposide operate by stabilizing TOP2ccs, ultimately generating genotoxic TOP2-DNA protein cross-links that require processing and repair. Here, we identify RAD54 like 2 (RAD54L2) as a factor promoting TOP2cc resolution. We demonstrate that RAD54L2 acts through a novel mechanism together with zinc finger protein associated with tyrosyl-DNA phosphodiesterase 2 (TDP2) and TOP2 (ZATT/ZNF451) and independent of TDP2. Our work suggests a model wherein RAD54L2 recognizes sumoylated TOP2 and, using its ATPase activity, promotes TOP2cc resolution and prevents DSB exposure. These findings suggest RAD54L2-mediated TOP2cc resolution as a potential mechanism for cancer therapy resistance and highlight RAD54L2 as an attractive candidate for drug discovery.

Establishment and Thorough Characterization of Xenograft (PDX) Models Derived from Patients with Pancreatic Cancer for Molecular Analyses and Chemosensitivity Testing.

Patient-derived xenograft (PDX) tumor models are essential for identifying new biomarkers, signaling pathways and novel targets, to better define key factors of therapy response and resistance mechanisms. Therefore, this study aimed at establishing pancreas carcinoma (PC) PDX models with thorough molecular characterization, and the identification of signatures defining responsiveness toward drug treatment. In total, 45 PC-PDXs were generated from 120 patient tumor specimens and the identity of PDX and corresponding patient tumors was validated. The majority of engrafted PDX models represent ductal adenocarcinomas (PDAC). The PDX growth characteristics were assessed, with great variations in doubling times (4 to 32 days). The mutational analyses revealed an individual mutational profile of the PDXs, predominantly showing alterations in the genes encoding KRAS, TP53, FAT1, KMT2D, MUC4, RNF213, ATR, MUC16, GNAS, RANBP2 and CDKN2A. Sensitivity of PDX toward standard of care (SoC) drugs gemcitabine, 5-fluorouracil, oxaliplatin and abraxane, and combinations thereof, revealed PDX models with sensitivity and resistance toward these treatments. We performed correlation analyses of drug sensitivity of these PDX models and their molecular profile to identify signatures for response and resistance. This study strongly supports the importance and value of PDX models for improvement in therapies of PC.

Editorial: Molecular and cellular mechanisms for cancer therapy resistance

Editorial: Molecular and cellular mechanisms for cancer therapy resistance

Acute Myeloid Leukemia (AML) from Older HSCs Is Resistant to Chemotherapy and Has an Altered DNA Damage Response

Acute myeloid leukemia (AML) is an aggressive malignancy that leads to the accumulation of immature myeloid precursors, resulting in progressive marrow failure and death. AML arises from the hematopoietic stem cell (HSC), either directly or through an early myeloid precursor. HSCs must survive for the entire human lifespan to provide the 200+ billion blood cells that are produced daily. They are subjected to the entirety of the aging process. Further, while AML is diagnosed in every age range, it disproportionately affects older patients, with a median age at diagnosis of 68. Older age, typically defined as 60 years of age or older, is a powerful, poor prognostic factor. Clinical trials show an estimated 5-year survival of less than 10% in these patients. Ten-year survival is even worse with one study estimating 2.4% of patients over 60 years of age alive at ten years when treated with chemotherapy. Importantly, this poor outcome is the result of disease that is significantly more resistant to therapy. Even though this clinical data is well known the underlying mechanisms of therapy resistance in older AML are unknown. To address this, we generated genetically identical AML cells from fetal, 12-week-old (Young) or 24-month-old (Old) murine HSCs. Retroviral vectors were used to express the oncogenic MLL-ENL fusion protein and NRas G12D. AML was confirmed by morphology, immunophenotype and all AML cells were lethal when injected into syngeneic recipients. Old AML cells demonstrated reduced autophagy, decreased NAD levels and impaired mitochondria consistent with a significant effect of biological aging. When treated with DNA damaging chemotherapy, Old AML cells were significantly more resistant than either Young or Fetal cells to both cytarabine and the anthracycline doxorubicin. To examine the DNA damage response AML cells were treated with doxorubicin and stained for the early DNA damage marker gH2AX. Old AML cells generated significantly more gH2AX than either Fetal or Young AML cells (Figure 1). We next examined the induction of p53 in response to treatment and in contrast to the gH2AX results Old AML cells induced the least amount of p53 in response to doxorubicin treatment (Figure 2). This data demonstrates that Old AML cells were more adept at recognizing DNA damage but least likely to induce p53 in response to it suggesting a difference in DNA repair. To better understand these differences RNA sequencing was performed and differentially expressed genes (DEGs) identified. When Fetal and Young AML cells were compared, no DEGs reached the false discovery threshold for significance, demonstrating that Fetal and Young AMLs have highly similar gene expression profiles. In contrast, when Old AML cells were compared to either Young or Fetal cells, hundreds of highly significant DEGs were identified, and KEGG pathway enrichment analysis revealed that several DNA repair pathways were significantly upregulated in Old AML. These include base excision repair (FDR p= 8.60E-06), mismatch repair (FDR p=3.26E-05), and nucleotide excision repair (FDR p= 0.00043). To further validate these pathways, the Beat AML TCGA dataset was assessed for the prognostic effect of increased expression of key genes in these pathways. Patients with increased expression of these genes had a trend toward worse survival (p=0.07) consistent with these pathways being clinically relevant. These data suggest that AML arising in older patients may overexpress DNA damage repair pathways leading to chemotherapy resistance and poor outcomes. Additional work on ways to increase sensitivity to Old AML cells is in progress.

Development of Circulating Tumor DNA (ctDNA) for Molecular Measurable Residual Disease (MRD) in Acute Myeloid Leukemia (AML)

Introduction: Despite advances in therapeutic options, the majority of AML patients experience relapsed or refractory disease. Standard-of-care (SOC) methods for MRD detection (multiparametric flow cytometry and single gene or structural variant molecular assays) are hampered by (1) inadequate lower limits of detection (LOD), (2) paucity of recurrent mutations across AML genomes, (3) insufficient numbers of identified mutations to characterize clonal evolution, and/or (4) need for invasive bone marrow biopsy. Herein we develop and apply non-invasive circulating tumor DNA (ctDNA) liquid biopsies using a customized Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) approach to simultaneously quantify MRD and characterize clonal dynamics in AML. Methods: To increase the number of tumor-specific mutations per case for MRD detection and improve the LOD for ctDNA-MRD, we performed whole exome sequencing (WES) on purified blasts and paired T cells (latter as germline control) from 24 patients with AML (n = 20 previously untreated, n = 4 with relapsed/refractory disease) and designed personalized hybrid capture panels. We combined the personalized panels with a shared hybrid capture panel of 58 recurrently mutated (‘canonical‘) AML genes covering 225kB as a composite approach to generate patient-specific AML-CAPP-Seq assays. We applied AML-CAPP-Seq to track single nucleotide variants (SNVs) in 468 biological samples from the 24 AML patients and 21 healthy control subjects. We compared the performance of MRD detection in cellular (bone marrow mononuclear cell/BMMC, peripheral blood mononuclear cell/PBMC) and cell-free (BM-ctDNA, PB-ctDNA) compartments against SOC clinical MRD assays as well as patient specific clinical outcomes. Results: We observed a median of 30 SNVs (IQR 16.5-40.25) per patient from WES, compared to only 2 (IQR 1-3.25) if using the AML-focused canonical panel alone (p < 0.0001) ( Fig. 1A). This number of variants enabled sensitive molecular disease monitoring in 96% (23/24) of patients with a LOD of < 0.01% tumor fraction from typical quantities of cell-free DNA. In contrast, when using the canonical panel alone, only 83% (20/24) of patients had 1 or more SNV to allow MRD detection. Notably, only 12.5% (3/24) of patients in this cohort had a feature amenable to SOC molecular MRD methods. Upon comparing quantitative levels of MRD in each biological compartment, we found that tumor burden in PB-ctDNA, BM-ctDNA and BMMC samples were highly correlated (r > 0.92, p < 0.0001) with similar amounts of disease. Interestingly, PB-ctDNA contained quantitatively more tumor DNA than corresponding PBMC samples (p = 0.0002), most pronounced at low tumor burdens (median 8.2-fold enrichment when mean tumor allele fraction in PBMCs was < 1%). These results suggest that PB-ctDNA effectively captures total body BM disease and mutational burden in AML and serves as a superior analyte for MRD monitoring compared to PBMCs. When evaluating the performance of AML-CAPP-Seq against clinical SOC methods, our assay correctly detected molecular disease in 99.6% (237/238) of samples known to contain measurable disease, including 98.2% (54/55) of samples at timepoints of clinical MRD positivity during clinical remission (CR). However, restricting trackable SNVs to only ‘canonical’ genes led to significant degradation of performance. Specifically, only 69.1% (38/55) of samples with positive MRD during CR by clinical SOC assay were detected by ctDNA if canonical genes alone were employed (p = 0.0001). Intriguingly, when using the complete gene panel, AML-CAPP-Seq also identified molecular MRD in 37.5% (30/80) of samples with undetectable MRD by SOC assays. Finally, stratifying patients who achieved CR after 1st treatment line based on PB-ctDNA MRD significantly predicted relapse free survival (p = 0.046, hazard ratio (HR) = 4.5). Moreover, attaining an MRD negative CR in PB-ctDNA at any timepoint was strongly prognostic of overall survival (p = 0.0004, HR = 8.5) ( Fig. 1B). Conclusions: ctDNA is a promising biomarker for ultrasensitive detection and tracking of MRD in AML without need for invasive bone marrow assessment. Tracking mutations beyond canonical AML genes improves performance of such assays. ctDNA analysis has numerous areas of clinical utility, including response and relapse prediction, evaluation of clonal dynamics, and exploration of mechanisms of therapy resistance.

The Mechanism of Therapy Resistance By Lineage Plasticity in AML and How to Overcome It

Acute myeloid leukemia (AML) is an aggressive blood cancer driven predominantly by mutations in epigenetic regulators. This characteristic allows AML to thrive in a hostile and rapidly changing environment where it needs to adapt to stressors both extrinsic (chemotherapy) and intrinsic (replicative and oxidative stress) to survive. Our study is the first to demonstrate the mechanism of how AML uses epigenetic reprogramming to adjust to these stressors via lineage plasticity. Venetoclax and Azacitidine (Ven/Aza) co-treatment is increasingly the front-line therapy for AML, functioning through BH3 inhibition and DNA hypomethylation. However, most patients will relapse with a highly aggressive refractory AML. Some studies suggest that monocytic character of the AML is responsible for relapse, but more recent evidence demonstrates that Ven/Aza treatment itself can induce a lineage switch to monocytic character. A potent selection mechanism facilitated by epigenetic dysregulation generating plastic clones is likely driving resistance, though the mechanism remains unclear and represents a significant barrier for AML treatment. Using a combination of single cell transcriptomics with surface immunophenotype sequencing in multiple human xenograft and primary patient samples, we discovered that this plasticity is widespread and mediated by epigenetic reprogramming of the tumor in direct response to the therapy. Broadly, we found that AML responds to Ven/Aza by transitioning into a reversible state of cell cycle arrest with transcriptional character similar to hematopoietic stem cells. This transient state protects AML from Azacitidine through replication arrest and protects from Venetoclax by upregulation of anti-apoptotic transcriptional programs. These protections operate simultaneously with reprogramming events driven by a switch from MYC-driven transcriptional programs to a PU.1-driven program. Reprogramming is both facilitated by and dependent on repression of the Chromatin Assembly Factor 1 (CAF1) complex, a chromatin-bound master epigenetic regulator responsible for maintaining lineage fidelity through transcriptional regulation via its role as a nucleosome assembly factor. Repressing CAF1 facilitates reprogramming where the output is an altered cell population that is distinct from the initial tumor both transcriptionally and by ATACseq. AML can repeatedly return to this plastic state, regardless of the number of cycles of Ven/Aza, and introducing other cytotoxic compounds does not remove this plastic stem-like population but instead initiates the iterative process of reprogramming anew until a different resistant population emerges. To find therapeutic vulnerabilities, we generated several PDX models of Ven/Aza resistance so that we could track the stages of acquired resistance in real time. When single cell data from these experiments were projected onto a reference map of normal hematopoiesis, we found that Ven/Aza resistance stems from a plastic population of cells that actively samples the niche by generating short-lived multi-lineage hybrid differentiated products. While the predominating output of this process was monocytic, we found that the induced plastic stem-like population was durably retained even after Ven/Aza withdrawal. To undermine this process, we took advantage of the plastic state to drive differentiation through an external stimulus. We expected this strategy to be particularly effective because CAF1 was repressed (CAF1 repression facilitates differentiation in every metazoan model) and, based on our single cell data, these plastic populations of AML cells express pro-differentiation surface receptors. In particular, Ven/Aza induced the expression of CSF3R (the G-CSF receptor) which when stimulated, drives neutrophil maturation through the JAK/STAT pathway. While G-CSF as a single agent or in sequence with Ven/Aza was unable to achieve maturation, when used concurrently with Ven/Aza (or recapitulated by CAF1 depletion using shRNA) we were able to drive neutrophil differentiation in vitro and fully resolve primary mouse and PDX tumors in vivo, even when using Ven/Aza resistant tumors as starting material. Together, these data demonstrate the possibility that Ven/Aza resistance can be completely subverted by taking advantage of the plastic state induced by the therapy to stimulate differentiation and resolve the disease.

An Updated Review on KRAS Mutation in Lung Cancer (NSCLC) and Its Effects on Human Health.

The largest cause of cancer-related fatalities worldwide is lung cancer. In its early stages, lung cancer often exhibits no signs or symptoms. Its signs and symptoms often appear when the condition is advanced. The Kirsten rat sarcoma virus oncogene homolog is one of the most frequently mutated oncogenes found in non-small cell lung cancer. Patients who have these mutations may do worse than those who do not, in terms of survival. To understand the nuances in order to choose the best treatment options for each patient, including combination therapy and potential resistance mechanisms, given the quick development of pharmaceuticals, it is necessary to know the factors that might contribute to this disease. It has been observed that single nucleotide polymorphisms altering let-7 micro-RNA might impact cancer propensity. On the other hand, gefitinib fails to stop the oncogenic protein from directly interacting with phosphoinositide3-kinase, which may explain its resistance towards cancer cells. Additionally, Atorvastatin may be able to overpower gefitinib resistance in these cancer cells that have this mutation regardless of the presence of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha. De novo lipogenesis is also regulated by this virus. To overcome these effects, several targeted therapies have been proposed. One such therapy is to use inhibitors of focal adhesion kinases. When this is inhibited, viral oncogene mutant cancers are effectively stopped because it functions downstream of the virus. Mutant oncoproteins like epidermal growth factor receptor may depend on Heat Shock protein90 chaperones more frequently than they do on natural counterparts that make it more attractive therapeutic target for this virus. Inhibition of the phosphoinositide 3-kinase pathway is frequent in lung cancer, and fabrication of inhibitors against this pathway can also be an effective therapeutic strategy. Blocking programmed cell death ligand1 is another therapy that may help T cells to recognize and eliminate cancerous cells. This homolog is a challenging therapeutic target due to its complex structural makeup and myriad biological characteristics. Thanks to the unrelenting efforts of medical research, with the use of some inhibitors, immunotherapy, and other combination methods, this problem is currently expected to be overcome.

Liquid Biopsy - A new diagnostic concept in oncology

The analysis of tumor cells circulating in the blood or of products of tumor cells circulating in other body fluids has gained increasing attention in recent years and is summarized under the term liquid biopsy (LB). LB includes the analysis of circulating tumor cells, cell-free circulating tumor-associated nucleic acids, extracellular vesicles, proteins, or other products that are released into the peripheral bloodstream by the primary or metastatic tumor. For a huge number of solid tumor entities, LB has already been successfully applied in preclinical and clinical studies for the detection, risk stratification, treatment monitoring and relapse detection. LB provides valuable real-time information on tumor cell development, therapeutic targets, and mechanisms of therapy resistance using a non-invasive peripheral blood test. In this article, the most important LB analytes and the current state of research are presented. In addition, the remaining obstacles and the diverse efforts to implement LB in clinical routine are critically discussed.

Characterizing the mutational burden, DNA methylation landscape, and proteome of germ cell tumor-related somatic-type malignancies to identify the tissue-of-origin, mechanisms of therapy resistance, and druggable targets

BackgroundGerm cell tumors (GCT) might undergo transformation into a somatic-type malignancy (STM), resulting in a cell fate switch to tumors usually found in somatic tissues, such as rhabdomyosarcomas or adenocarcinomas. STM is associated with a poor prognosis, but the molecular and epigenetic mechanisms triggering STM are still enigmatic, the tissue-of-origin is under debate and biomarkers are lacking.MethodsTo address these questions, we characterized a unique cohort of STM tissues on mutational, epigenetic and protein level using modern and high-throughput methods like TSO assays, 850k DNA methylation arrays and mass spectrometry.Results and conclusionsFor the first time, we show that based on DNA methylation and proteome data carcinoma-related STM more closely resemble yolk-sac tumors, while sarcoma-related STM resemble teratoma. STM harbor mutations in FGF signaling factors (FGF6/23, FGFR1/4) highlighting the corresponding pathway as a therapeutic target. Furthermore, STM utilize signaling pathways, like AKT, FGF, MAPK, and WNT to mediate molecular functions coping with oxidative stress, toxin transport, DNA helicase activity, apoptosis and the cell cycle. Collectively, these data might explain the high therapy resistance of STM. Finally, we identified putative novel biomarkers secreted by STM, like EFEMP1, MIF, and DNA methylation at specific CpG dinucleotides.