Mechanisms Of Therapy Resistance Research Articles

Identification and characterization of chemotherapy resistant high-risk neuroblastoma persister cells.

Relapse rates in high-risk neuroblastoma remain exceedingly high. The malignant cells that are responsible for relapse have not been identified, and mechanisms of therapy resistance remain poorly understood. Here, we used single nucleus RNA sequencing and bulk whole genome sequencing to identify and characterize the residual malignant persister cells that survive chemotherapy from a cohort of 20 matched diagnosis and definitive surgery tumor samples from patients treated with high-risk neuroblastoma induction chemotherapy. We show that persister cells share common mechanisms of chemotherapy escape including suppression of MYCN activity and activation of NF-κB signaling, the latter is further enhanced by cell-cell communication between the malignant cells and the tumor microenvironment. Overall, our work dissects the transcriptional landscape of cellular persistence in high-risk neuroblastoma and paves the way to the development of new therapeutic strategies to prevent disease relapse.

Determinants of resistance and response to melanoma therapy.

Metastatic melanoma is among the most enigmatic advanced cancers to clinically manage despite immense progress in the way of available therapeutic options and historic decreases in the melanoma mortality rate. Most patients with metastatic melanoma treated with modern targeted therapies (for example, BRAFV600E/K inhibitors) and/or immune checkpoint blockade (for example, anti-programmed death 1 therapy) will progress, owing to profound tumor cell plasticity fueled by genetic and nongenetic mechanisms and dichotomous host microenvironmental influences. Here we discuss the determinants of tumor heterogeneity, mechanisms of therapy resistance and effective therapy regimens that hold curative promise.

Utility of Targeted Sequencing Compared to FISH for Detection of Chronic Lymphocytic Leukemia Copy Number Alterations.

Chronic lymphocytic leukemia (CLL) is characterized by multiple copy number alterations (CNAs) and somatic mutations that are central to disease prognosis, risk stratification, and mechanisms of therapy resistance. Fluorescence in situ hybridization (FISH) panels are widely used in clinical applications as the gold standard for screening prognostic chromosomal abnormalities in CLL. DNA sequencing is an alternative approach to identifying CNAs but is not an established method for clinical CNA screening. We sequenced DNA from 509 individuals with CLL or monoclonal B-cell lymphocytosis (MBL), the precursor to CLL, using a targeted sequencing panel of 59 recurrently mutated genes in CLL and additional amplicons across regions affected by clinically relevant CNAs [i.e., del(17p), del(11q), del(13q), and trisomy 12]. We used the PatternCNV algorithm to call CNA and compared the concordance of calling clinically relevant CNAs by targeted sequencing to that of FISH. We found a high accuracy of calling CNAs via sequencing compared to FISH. With FISH as the gold standard, the specificity of targeted sequencing was >95%, sensitivity was >86%, positive predictive value was >90%, and negative predictive value was >84% across the clinically relevant CNAs. Using targeted sequencing, we were also able to identify other common CLL-associated CNAs, including del(6q), del(14q), and gain 8q, as well as complex karyotype, defined as the presence of 3 or more chromosomal abnormalities, in 26 patients. In a single and cost-effective assay that can be performed on stored DNA samples, targeted sequencing can simultaneously detect CNAs, somatic mutations, and complex karyotypes, which are all important prognostic features in CLL.

Peroxiporins in Triple-Negative Breast Cancer: Biomarker Potential and Therapeutic Perspectives.

Triple-negative breast cancer (TNBC) remains one of the most challenging subtypes since it is initially characterized by the absence of specific biomarkers and corresponding targeted therapies. Advances in methodology, translational informatics, genomics, and proteomics have significantly contributed to the identification of therapeutic targets. The development of innovative treatments, such as antibody-drug conjugates and immune checkpoint inhibitors, alongside chemotherapy, has now become the standard of care. However, the quest for biomarkers defining therapy outcomes is still ongoing. Peroxiporins, which comprise a subgroup of aquaporins, which are membrane pores facilitating the transport of water, glycerol, and hydrogen peroxide, have emerged as potential biomarkers for therapy response. Research on peroxiporins reveals their involvement beyond traditional channeling activities, which is also reflected in their cellular localization and roles in cellular signaling pathways. This research on peroxiporins provides fresh insights into the mechanisms of therapy resistance in tumors, offering potential avenues for predicting treatment outcomes and tailoring successful TNBC therapies.

The Role of Nrf2 in the Regulation of Mitochondrial Function and Ferroptosis in Pancreatic Cancer.

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) represents the master regulator of the cellular antioxidant response and plays a critical role in tumorigenesis. This includes a preventive effect of Nrf2 on cell death through ferroptosis, which represents an essential mechanism of therapy resistance in malignant tumors, such as pancreatic ductal adenocarcinoma (PDAC) as one of the most aggressive and still incurable tumors. Addressing this issue, we provide an overview on Nrf2 mediated antioxidant response with particular emphasis on its effect on mitochondria as the organelle responsible for the execution of ferroptosis. We further outline how deregulated Nrf2 adds to the progression and therapy resistance of PDAC, especially with respect to the role of ferroptosis in anti-cancer drug mediated cell killing and how this is impaired by Nrf2 as an essential mechanism of drug resistance. Our review further discusses recent approaches for Nrf2 inhibition by natural and synthetic compounds to overcome drug resistance based on enhanced ferroptosis. Finally, we provide an outlook on therapeutic strategies based on Nrf2 inhibition combined with ferroptosis inducing drugs.

Graphene-Based Photodynamic Therapy and Overcoming Cancer Resistance Mechanisms: A Comprehensive Review.

Photodynamic therapy (PDT) is a non-invasive therapy that has made significant progress in treating different diseases, including cancer, by utilizing new nanotechnology products such as graphene and its derivatives. Graphene-based materials have large surface area and photothermal effects thereby making them suitable candidates for PDT or photo-active drug carriers. The remarkable photophysical properties of graphene derivates facilitate the efficient generation of reactive oxygen species (ROS) upon light irradiation, which destroys cancer cells. Surface functionalization of graphene and its materials can also enhance their biocompatibility and anticancer activity. The paper delves into the distinct roles played by graphene-based materials in PDT such as photosensitizers (PS) and drug carriers while at the same time considers how these materials could be used to circumvent cancer resistance. This will provide readers with an extensive discussion of various pathways contributing to PDT inefficiency. Consequently, this comprehensive review underscores the vital roles that graphene and its derivatives may play in emerging PDT strategies for cancer treatment and other medical purposes. With a better comprehension of the current state of research and the existing challenges, the integration of graphene-based materials in PDT holds great promise for developing targeted, effective, and personalized cancer treatments.

BRD4 degraders may effectively counteract therapeutic resistance of leukemic stem cells in AML and ALL.

Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are life-threatening hematopoietic malignancies characterized by clonal expansion of leukemic blasts in the bone marrow and peripheral blood. The epigenetic reader BRD4 and its downstream effector MYC have recently been identified as potential drug targets in human AML and ALL. We compared anti-leukemic efficacies of the small-molecule BET inhibitor JQ1 and the recently developed BRD4 degraders dBET1 and dBET6 in AML and ALL cells. JQ1, dBET1, and dBET6 were found to suppress growth and viability in all AML and ALL cell lines examined as well as in primary patient-derived AML and ALL cells, including CD34+/CD38- and CD34+/CD38+ leukemic stem and progenitor cells, independent of the type (variant) of leukemia or molecular driver expressed in leukemic cells. Moreover, we found that dBET6 overcomes osteoblast-induced drug resistance in AML and ALL cells, regardless of the type of leukemia or the drug applied. Most promising cooperative or even synergistic drug combination effects were seen with dBET6 and the FLT3 ITD blocker gilteritinib in FLT3 ITD-mutated AML cells, and with dBET6 and the multi-kinase blocker ponatinib in BCR::ABL1+ ALL cells. Finally, all BRD4-targeting drugs suppressed interferon-gamma- and tumor necrosis factor-alpha-induced expression of the resistance-related checkpoint antigen PD-L1 in AML and ALL cells, including LSC. In all assays examined, the BRD4 degrader dBET6 was a superior anti-leukemic drug compared with dBET1 and JQ1. Together, BRD4 degraders may provide enhanced inhibition of multiple mechanisms of therapy resistance in AML and ALL.

PML restrains p53 activity and cellular senescence in clear cell renal cell carcinoma

Clear-cell renal cell carcinoma (ccRCC), the major subtype of RCC, is frequently diagnosed at late/metastatic stage with 13% 5-year disease-free survival. Functional inactivation of the wild-type p53 protein is implicated in ccRCC therapy resistance, but the detailed mechanisms of p53 malfunction are still poorly characterized. Thus, a better understanding of the mechanisms of disease progression and therapy resistance is required. Here, we report a novel ccRCC dependence on the promyelocytic leukemia (PML) protein. We show that PML is overexpressed in ccRCC and that PML depletion inhibits cell proliferation and relieves pathologic features of anaplastic disease in vivo. Mechanistically, PML loss unleashed p53-dependent cellular senescence thus depicting a novel regulatory axis to limit p53 activity and senescence in ccRCC. Treatment with the FDA-approved PML inhibitor arsenic trioxide induced PML degradation and p53 accumulation and inhibited ccRCC expansion in vitro and in vivo. Therefore, by defining non-oncogene addiction to the PML gene, our work uncovers a novel ccRCC vulnerability and lays the foundation for repurposing an available pharmacological intervention to restore p53 function and chemosensitivity.

Abstract PS17-04: Clinical and Genomic Features of ER-Positive/HER2-negative Metastatic Breast Cancer in AURORA Molecular Screening Initiative (BIG 14-01): Mechanisms of Endocrine Therapy Resistance and Implications for Adjuvant Approaches

Abstract Background: Up to 30% of patients (pts) with ER-positive early breast cancers develop metastatic relapse. The molecular differences between endocrine therapy (ET)-sensitive and ET-resistant relapses, as well as the impact of specific adjuvant ET-based therapies, remain unclear. Methods: The AURORA program (NCT02102165) analyzed multi-omics data of paired primary (prim) and metastatic (meta) tumor tissue, along with plasma samples, from 1,156 patients with metastatic breast cancer (MBC). Targeted genome sequencing (TGS), RNA sequencing, and circulating tumor DNA analysis were performed. ET-resistance at MBC diagnosis was defined following the 5th ESO-ESMO ABC Guidelines. Results: We studied 628 pts with metastatic ER+/HER2- disease. Patient´s median age was 56 years, 133 (21%) were premenopausal, 314 (50%) had ET-resistance at recurrence (9% primary, 41% secondary), 132 (21%) had ET-sensitive recurrence, and the rest were ET-naive recurrences or de novo MBC. Adjuvant treatment was aromatase inhibitor (AI) +/- ovarian function suppression (OFS) in 288 (46%) pts, while 159 (25%) had tamoxifen only (+/- OFS). Gene expression correlation significantly differed (p < 0.005) based on ET-resistance in 92 paired samples. ET-sensitive (n=11) and de-novo tumors (n=41) showed higher correlation than primary (n=7) and secondary ET-resistance (n=32), regardless of adjuvant ET type. Prim and meta showed concordance in 91% of IHC subtypes and 62% of intrinsic subtypes. Intrinsic subtype showed 9% of prim luminal tumors switching to meta non-luminal, with 66% of Luminal A switching to Luminal B. Neither IHC nor intrinsic subtype switching was associated with the type of adjuvant ET or ET resistance. Pts with meta non-luminal intrinsic subtype (18%) had worse PFS on CDK4/6i (HR 4.0, 95% CI 1.9-8.7) and OS (HR 3.6, 95% CI 2.0-5.7) than luminal subtypes. TGS was performed in 534 meta (365 before 1L, 305 pairs). In meta before 1L, the dN/dS algorithm revealed selection in 17 genes, including TP53, PIK3CA, and ESR1. Mutations (mut) in ESR1, ERBB2, ERBB3, and RB1 among others, were specifically identified in meta. The incidence of ESR1mut was 3% in prim, 12.6% before 1L, 23.4% after 1L, and 4.7% in meta from ET-naive tumors (Table). Before 1L, ESR1mut was higher after adjuvant AI vs tamoxifen (21% vs. 4%, p< 0.001). ESR1mut incidence varied with ET-resistance (16% primary, 19% secondary, 9% sensitive, 4% ET-naïve/de novo, p< 0.01). Paired samples showed that ESR1mut were mainly acquired events and associated with higher ER mRNA signaling (hallmark estrogen response) compared to ESR1wt (p < 0.05). The agreement between ESR1mut detected in ctDNA and tissue was high before 1L (90%), and slightly lower after 1L (84%). In a multivariate model with relevant clinical factors, ESR1mut were associated with worse OS (HR 1.76, 95% CI 1.2-2.5, p=0.003), independently of TP53 and PIK3CA mutational status. Conclusion: AURORA study sheds light on metastatic tumor alterations acquired under anti-cancer therapy, in ER+/HER2- MBC. We observed a high prevalence of acquired ESR1mut prior to the initiation of first-line therapy, particularly in tumors exposed to adjuvant AI or with primary or secondary ET_resistance. The association of ESR1muts with poorer OS underscores the importance of implementing effective adjuvant ET strategies to prevent the emergence of these mutations. Frequency of driver gene mutations in primary and metastastic tumors before 1st-line treatment according to adjuvant ET and type of ET resistance Table. Citation Format: Angel Guerrero-Zotano, Matteo Benelli, Alexandre Irrthum, David Cameron, Lorenzo Ferrando, Dario Romagnoli, Marta Paoli, Arnau Llinas, Maya Dadiani, Danai Fimereli, Mafalda Oliveira, Carmela Caballero, Thayane Crestani, Elisa Agostinetto, Diogo Martins-Branco, Florentine Hilbers, Einav Gal-Yam, Marija Balic, Fatima Cardoso, Jorge Reis-Filho, Christos Sotiriou, Giuseppe Curigliano, Barbro Linderholm, Evandro de Azambuja, Susan Knox, Cristina Rotaru, Eva Ciruelos, Nancy E Davidson, Giuseppe Viale, Donatienne Taylor, Jean-Luc Canon, Lourdes Calvo, Joan Albanell, Marco Colleoni, Caroline Duhem, Nadia Harbeck, Catherine Herremans, Sibylle Loibl, Sandrine Marreaud, Elsemieke Scheepers, Alice Raimbault, Theodora Goulioti, Andrea Vingiani, Chiara Biagioni, Sebastian Vosberg, Gabriele Zoppoli, Jose Seoane, David Venet, Philippe Aftimos, Martine Piccart. Clinical and Genomic Features of ER-Positive/HER2-negative Metastatic Breast Cancer in AURORA Molecular Screening Initiative (BIG 14-01): Mechanisms of Endocrine Therapy Resistance and Implications for Adjuvant Approaches [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS17-04.

Loss of the stress sensor GADD45A promotes stem cell activity and ferroptosis resistance in LGR4/HOXA9-dependent AML

AbstractThe overall prognosis of acute myeloid leukemia (AML) remains dismal, largely because of the inability of current therapies to kill leukemia stem cells (LSCs) with intrinsic resistance. Loss of the stress sensor growth arrest and DNA damage-inducible 45 alpha (GADD45A) is implicated in poor clinical outcomes, but its role in LSCs and AML pathogenesis is unknown. Here, we define GADD45A as a key downstream target of G protein-coupled receptor (LGR)4 pathway and discover a regulatory role for GADD45A loss in promoting leukemia-initiating activity and oxidative resistance in LGR4/HOXA9-dependent AML, a poor prognosis subset of leukemia. Knockout of GADD45A enhances AML progression in murine and patient-derived xenograft (PDX) mouse models. Deletion of GADD45A induces substantial mutations, increases LSC self-renewal and stemness in vivo, and reduces levels of reactive oxygen species (ROS), accompanied by a decreased response to ROS-associated genotoxic agents (eg, ferroptosis inducer RSL3) and acquisition of an increasingly aggressive phenotype on serial transplantation in mice. Our single-cell cellular indexing of transcriptomes and epitopes by sequencing analysis on patient-derived LSCs in PDX mice and subsequent functional studies in murine LSCs and primary AML patient cells show that loss of GADD45A is associated with resistance to ferroptosis (an iron-dependent oxidative cell death caused by ROS accumulation) through aberrant activation of antioxidant pathways related to iron and ROS detoxification, such as FTH1 and PRDX1, upregulation of which correlates with unfavorable outcomes in patients with AML. These results reveal a therapy resistance mechanism contributing to poor prognosis and support a role for GADD45A loss as a critical step for leukemia-initiating activity and as a target to overcome resistance in aggressive leukemia.

Abstract LB334: Mechanisms of endocrine resistance to letrozole, fulvestrant, and palbociclib are associated with increased sensitivity to hexyl-cuban-1-yl biguanide inhibitors of CYP3A4 mediated epoxyeicosatrienoic acid biosynthesis

Abstract Introduction: Eicosanoid mediated mechanisms of hormone therapy resistance in ER+HER2- breast cancer are poorly understood. Cytochrome P450 arachidonic acid (AA) epoxygenase-derived epoxyeicosatrienoic acids (EETs) contribute to breast cancer progression by promoting mitochondrial oxidative phosphorylation (OXPHOS). However, it remains unclear how EETs contribute to hormonal therapy resistance. In this study, the well-known letrozole resistant (LR) MCF-7 AC1 cell line overexpressing aromatase was selected for fulvestrant resistance (FR). A clonal cell line resistant to letrozole and fulvestrant (LR/FR) also exhibited resistance to palbociclib (PR) (MCF-7 AC1 LR/FR/PR). Xenograft tumors of this cell line were resistant to letrozole, fulvestrant, and palbociclib. Structure activity studies and modeling led to the development of CYP3A4 AA epoxygenase inhibiting biguanides hexyl cuban-1-yl biguanide (HCB) and fluorinated hexyl derivatives C5F2-HCB and C6F3-HCB. Fluorination on the hexyl moiety led to broader inhibition of EET regioisomer biosynthesis. These agents were tested for inhibition of proliferation, signaling, and OXPHOS/glycolysis balance in the MCF-7 AC1 LR/FR/PR cell line. Results: In the MCF-7 AC1 LR/FR/PR clonal cell line cyclin D1 and estrogen receptor (ER) expression were undetectable, while cyclin E1, CDK4, CDK6, CYP3A4, and c-MYC were upregulated 11, 40, 13, 10, and 12- fold (n=3, p<0.001) compared to the MCF-7 cell line. LC-MS analysis demonstrated 1.8, 1.4, and 1.2- fold higher total cellular levels of (±)8,9, (±)11,12, and (±)14,15-EET in the MCF-7 AC1 LR/FR/PR cell line compared to the MCF-7 cell line (n=3, p<0.05). C6F3-HCB suppressed cellular levels of EETs: (±)8,9-EET by 55% (n=3, p<0.031), (±)11,12-EET by 53% (n=3, p<0.029), and (±)14,15-EET by 60% (n=3, P<0.0001). The MCF-7 AC1 LR/FR/PR cell line was more potently inhibited by hexyl-cuban-1-yl biguanides compared to the MCF-7 cell line, exhibiting lower IC50 values (MCF-7 AC1 LR/FR/PR IC50 vs. MCF-7 IC50; HCB 4.1 vs. 7.0 uM; for C5F2-HCB 10 vs. 25 uM; C6F3-HCB: 5.4 vs. 12 uM; P values all <0.05). Measurement of spare respiratory capacity revealed that the MCF-7 AC1 LR/FR/PR cell line had 4.2% spare respiratory capacity compared to 34% for MCF-7, indicating a lower OXPHOS reserve for the resistant cells. Additionally, the MCF-7 AC1 LR/FR/PR cell line displayed a 2.4-fold higher extracellular acidification rate (ECAR) than the MCF-7 cell line, indicating a higher glycolysis rate (Warburg effect) in the resistant cells. Conclusion: Selection for fulvestrant resistance of MCF-7 AC1 cells resulted in upregulation of CYP3A4 and biosynthesis of hormone therapy resistance associated EETs while correlating with greater sensitivity to fluorinated hexyl-cuban-1-yl biguanides that inhibit EET biosynthesis. Citation Format: Zhijun Guo, Jianxun Lei, Allison Makovec, Swaathi Jayaraman, John R. Hawse, Carol Lange, Elizabeth Ambrose, Gunda I. Georg, Tony D'Assoro, Goetz P. Matthew, David A. Potter. Mechanisms of endocrine resistance to letrozole, fulvestrant, and palbociclib are associated with increased sensitivity to hexyl-cuban-1-yl biguanide inhibitors of CYP3A4 mediated epoxyeicosatrienoic acid biosynthesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB334.

The senescence journey in cancer immunoediting.

Cancer progression is continuously controlled by the immune system which can identify and destroy nascent tumor cells or inhibit metastatic spreading. However, the immune system and its deregulated activity in the tumor microenvironment can also promote tumor progression favoring the outgrowth of cancers capable of escaping immune control, in a process termed cancer immunoediting. This process, which has been classified into three phases, i.e. "elimination", "equilibrium" and "escape", is influenced by several cancer- and microenvironment-dependent factors. Senescence is a cellular program primed by cells in response to different pathophysiological stimuli, which is based on long-lasting cell cycle arrest and the secretion of numerous bioactive and inflammatory molecules. Because of this, cellular senescence is a potent immunomodulatory factor promptly recruiting immune cells and actively promoting tissue remodeling. In the context of cancer, these functions can lead to both cancer immunosurveillance and immunosuppression. In this review, the authors will discuss the role of senescence in cancer immunoediting, highlighting its context- and timing-dependent effects on the different three phases, describing how senescent cells promote immune cell recruitment for cancer cell elimination or sustain tumor microenvironment inflammation for immune escape. A potential contribution of senescent cells in cancer dormancy, as a mechanism of therapy resistance and cancer relapse, will be discussed with the final objective to unravel the immunotherapeutic implications of senescence modulation in cancer.

Abstract 527: Cancer cells in the resistant, endocycling cell state exhibit centrosome amplification

Abstract Therapy resistance is one of the most common underlying causes of poor prognosis in cancer patients, ultimately driving fatal outcome. We have previous demonstrated that following chemotherapeutic stress, a subset of cancer cells undergo a mitotic skip and enter an endocycle, causing whole-genome duplication without division. This endocycling cell state is resistant to cytotoxic therapies and thus is an actuator of therapy resistance. Cells in the endocycling state eventually undergo depolyploidization and repopulate the tumor, observed clinically as a recurrence. We also demonstrated that the minus-end directed motor protein KIFC1, a mediator of centrosome clustering, has higher expression in cells following treatment. Centrosome amplification (CA) involves aberrations in centrosome number, where cells have 3 centrosomes. Cells with extra centrosomes create a multi-polar spindle during mitosis, which presents high risk for cell death and arrest, but cancer cells with CA avoid this by clustering extra centrosomes to create a pseudo-bipolar spindle. Based upon this data, we hypothesize that studying centrosome dynamics and abnormalities presents a window into better understanding endocycle biology and this novel mechanism of therapy resistance. Cancer cells were treated with LD50 dose of cisplatin and released from treatment for varying lengths of time. CA in endocycling cells increased from 59.2% to 100% during the treatment recovery period while maintaining identical levels of clustering. Endocycling cancer cells have a nearly 5-fold increase in declustered centrosomes compared to proliferative cells. Centrosomes in endocycling cells are displaced from the perinuclear region and toward the cell periphery. We hypothesize that centrosome number may indicate the number of times a cell has endocycled and provide insight into the timing and mechanism of mitotic bypass. Centrosomes play a major role in microtubule nucleation, affecting cell motility and polarity, and act as signaling platforms, enhancing signaling activation and specificity. Because of this, we hypothesize that CA may be a critical mechanism for endocycling cancer cells to survive and maintain this resistant cell state. Citation Format: Madison Purkerson, Sarah Amend, Kenneth J. Pienta. Cancer cells in the resistant, endocycling cell state exhibit centrosome amplification [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 527.

Abstract 6612: Therapy-resistant endocycling cancer cells alter mitochondrial structure and metabolism to survive therapeutic stress

Abstract Therapy resistance is responsible for 90% of cancer-related deaths. We identified a novel mechanism of therapy resistance in which cancer cells enter an endocycling cell state, undergoing repeated S and G phases without dividing. As a result, resistant endocycling cells are 40 times larger than mitotic cancer cell controls. Interestingly, we observed that these non-proliferative endocycling cells can eventually give rise to proliferative progeny weeks after chemotherapy is removed, modeling cancer recurrence. We hypothesize that this endocycling cancer cell state is a critical driver of therapy resistance and cancer lethality. We evaluated oxidative stress in endocycling cells that survived the days and weeks following chemotherapy treatment using DCF-DA staining. We found increased levels of reactive oxygen species (ROS) in endocycling prostate cancer cells compared to untreated mitotic cells when normalized to cell size. Given that mitochondria are major producers and regulators of ROS, we hypothesize that cells surviving in this resistant state alter mitochondrial structure and function in response to increased oxidative stress. We performed immunofluorescence and confocal microscopy on mitochondria in untreated cells and cells following chemotherapy treatment to observe their morphology. Surviving endocycling cells had increased mitochondrial fragmentation when compared to the untreated group, a result that was further supported by reduced expression of phospho-DRP1 Ser637 via western blot. To assess changes in glucose metabolism, we then utilized isotope tracing-based metabolomics with [U-13C]glucose on untreated and surviving endocycling cells following chemotherapy. Endocycling cells had decreased labeling of 13C in TCA cycle metabolites, suggesting a decrease in mitochondrial function when compared to untreated cells. Ongoing work involves further studying metabolic reprogramming as cells enter this resistant state with parallel labeling experiments using [1,2-13C]glucose and [U-13C]glutamine to perform mathematical modeling-based 13C-metabolic flux analysis. In addition, we engineered a cell line to track mitochondria in real-time to quantify mitochondrial dynamics underlying the morphology observed in untreated and surviving cells. Our goal is to characterize how this therapy-resistant cell state alters its mitochondrial structure and function to survive therapeutic stress. Identifying vulnerabilities of this phenotype will enable new approaches for eliminating drug resistance in cancer. Citation Format: Melvin Li, Sarah R. Amend, Kenneth J. Pienta. Therapy-resistant endocycling cancer cells alter mitochondrial structure and metabolism to survive therapeutic stress [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 6612.

Abstract 5592: HMGA1 modulates chromatin state and transcriptional networks involved in plasticity in refractory myeloid leukemia

Abstract Although High Mobility Group A1 (HMGA1) chromatin regulators are overexpressed in refractory acute myeloid leukemia (AML) and diverse solid tumors, targetable mechanisms underlying HMGA1 remain elusive. HMGA1 is highly expressed in stem cells and poorly differentiated cancers where it modulates chromatin structure to activate developmental gene networks. In AML arising from JAK2V617F myeloproliferative neoplasms (MPN), HMGA1 is required for leukemogenesis by inducing transcriptional networks that drive aberrant proliferation and cell fate. Like MPN AML, KMT2A-rearranged (KMT2A-r) AML is refractory to therapy and highly lethal. KMT2A-r AML is caused by rearrangements of the KMT2A gene, which encode abnormal fusion proteins that induce pro-leukemogenic genes, including HOX genes. While prior work identified KMT2A fusion partners and protein complexes, this has not led to better therapies for most patients. We therefore sought to examine HMGA1 function in KMT2A-r AML. We hypothesized that: 1) HMGA1 drives plasticity in KMT2A-r AML by altering chromatin state and transcriptional networks, and, 2) Targeting HMGA1 will enhance sensitivity to therapy. In two KMT2A-r AML patient cohorts, HMGA1 is highly overexpressed compared to CD34+ stem and progenitors from healthy controls, suggesting it drives leukemogenesis. To define HMGA1 function, we silenced HMGA1 using CRISPR or short hairpin RNA in KMT2A-r cell lines (MOLM-14, THP-1, MV4-11). Strikingly, silencing HMGA1 disrupts proliferation and clonogenicity in vitro, while prolonging survival in immunosuppressed mice transplanted with KMT2A-r AML cells. Intriguingly, leukemic cells that engraft in the bone marrow from the pool of cells with HMGA1 silencing express higher levels of HMGA1, suggesting that escape from gene silencing and a specific level of HMGA1 is required for leukemic engraftment and expansion. HMGA1 also increases in KMT2A-r AML cells that become resistant to cytarabine (AraC), suggesting that HMGA1 endows AML cells with the capacity to survive and expand after AraC. Because repression of tumor suppressors was recently defined as a mechanism of therapy resistance in AML, we also examined tumor suppressor pathways in both JAK2V617F and KMT2A-r AML. Intriguingly, HMGA1 occupies the promoter region of the CDKN1A locus by chromatin immunoprecipitation and represses its expression in KMT2A-r and JAK2V617F AML cells. To define additional mechanisms underlying HMGA1 in KMT2A-r AML, multi-omics studies are underway. In preliminary work thus far, we also found that HMGA1 induces HOX genes. Together, these findings implicate HMGA1 as a promising therapeutic target and novel epigenetic regulator in KMT2A-r AML by altering chromatin state and downstream gene expression. Citation Format: Bailey E. West, Jung-Hyun Kim, Audrey-Ann Supreme, Iliana Herrera, Zanshé Thompson, Li Z. Luo, Faiza Shaik, Joseph Kim, Hyunsung Woo, Soheil Meshinchi, Rhonda E. Ries, Linda M. Resar. HMGA1 modulates chromatin state and transcriptional networks involved in plasticity in refractory myeloid leukemia [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 5592.

Abstract 6925: Integrative single-cell tracking of genome evolution and tumor cell plasticity in small cell lung cancer (SCLC)

Abstract SCLC is the deadliest type of lung cancer, which is mostly diagnosed at late stages and accounts for 15% of pulmonary tumors. Even though chemotherapy is initially effective, patients with SCLC relapse quickly and develop resistant tumors. Genome studies revealed a universal loss of TP53 and RB1; recent transcriptional studies categorized SCLC based on the expression of 4 lineage transcription factors (TFs). We aimed to dissect how genomic cues impact transcriptional phenotypes, tumor cell plasticity and the dynamics of tumor and immune cell interactions, to thus decipher molecular mechanisms of phenotypic divergence and therapy resistance in SCLC. We performed genome sequencing and single cell transcriptome profiling of 55 tumors from patients with SCLC, including primary tumors, local and distant metastases and of paired relapsing tumors acquired after chemotherapy. Single cell sequencing was performed with the 10x Genomics platform and yielded > 200.000 cells. Transcriptome profiles at single cell levels revealed co-expression of at least two of the lineage TFs ASCL1, NEUROD1 or POU2F3 in each tumor. Copy number (cn) estimation for transcriptome data pointed to genomic events impacting transcriptional heterogeneity within the tumor. Furthermore, our data pointed to a marked intra- and interpatient, as well as to inter-metastatic heterogeneity. To specifically investigate how underlying genetic alterations affect molecular phenotypes, integrative studies across all patients were performed. This revealed shared transcriptional programs in tumors with amplifications of MYC family members; trajectory inference for cases with low and high-level copy gains of MYCN pointed to distinct transcriptional states. We performed multi-regional and longitudinal studies of matched patient cases to reconstruct the genomic patterns of clonal evolution, and projected genomic subsets to transcriptional profiles at single cell level, thus analyzing the effect of genome diversity on the transcriptional landscape. Additionally, tumor intrinsic profiles of heterogeneity were mapped to single cell data of patient tumors to elucidate the effects on tumor-immune cell interactions. All together our data points to a concerted regulation of a multitude of lineage factors and transcriptional programs in each SCLC tumor and provides a first comprehensive framework for the study of underlying genetic alterations that shape the transcriptional landscape and phenotypic plasticity in SCLC. Citation Format: Marcel Schmiel, Laura Kaiser, Maria Cartolano, Martin Peifer, Roman K. Thomas, Julie George. Integrative single-cell tracking of genome evolution and tumor cell plasticity in small cell lung cancer (SCLC) [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 6925.

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.