Promotes Cancer Cell Survival Research Articles

Overcoming Chemoresistance in Cancer: The Promise of Crizotinib.

Chemoresistance is a major obstacle in cancer treatment, often leading to disease progression and poor outcomes. It arises through various mechanisms such as genetic mutations, drug efflux pumps, enhanced DNA repair, and changes in the tumor microenvironment. These processes allow cancer cells to survive despite chemotherapy, underscoring the need for new strategies to overcome resistance and improve treatment efficacy. Crizotinib, a first-generation multi-target kinase inhibitor, is approved by the FDA for the treatment of ALK-positive or ROS1-positive non-small cell lung cancer (NSCLC), refractory inflammatory (ALK)-positive myofibroblastic tumors (IMTs) and relapsed/refractory ALK-positive anaplastic large cell lymphoma (ALCL). Crizotinib exists in two enantiomeric forms: (R)-crizotinib and its mirror image, (S)-crizotinib. It is assumed that the R-isomer is responsible for the carrying out various processes reviewed here The S-isomer, on the other hand, shows a strong inhibition of MTH1, an enzyme important for DNA repair mechanisms. Studies have shown that crizotinib is an effective multi-kinase inhibitor targeting various kinases such as c-Met, native/T315I Bcr/Abl, and JAK2. Its mechanism of action involves the competitive inhibition of ATP binding and allosteric inhibition, particularly at Bcr/Abl. Crizotinib showed synergistic effects when combined with the poly ADP ribose polymerase inhibitor (PARP), especially in ovarian cancer harboring BRCA gene mutations. In addition, crizotinib targets a critical vulnerability in many p53-mutated cancers. Unlike its wild-type counterpart, the p53 mutant promotes cancer cell survival. Crizotinib can cause the degradation of the p53 mutant, sensitizing these cancer cells to DNA-damaging substances and triggering apoptosis. Interestingly, other reports demonstrated that crizotinib exhibits anti-bacterial activity, targeting Gram-positive bacteria. Also, it is active against drug-resistant strains. In summary, crizotinib exerts anti-tumor effects through several mechanisms, including the inhibition of kinases and the restoration of drug sensitivity. The potential of crizotinib in combination therapies is emphasized, particularly in cancers with a high prevalence of the p53 mutant, such as triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSOC).

The novel SMYD3 inhibitor EM127 impairs DNA repair response to chemotherapy-induced DNA damage and reverses cancer chemoresistance

BackgroundSMYD3 has been found implicated in cancer progression. Its overexpression correlates with cancer growth and invasion, especially in gastrointestinal tumors. SMYD3 transactivates multiple oncogenic mechanisms, favoring cancer development. Moreover, it was recently shown that SMYD3 is required for DNA restoration by promoting homologous recombination (HR) repair.MethodsIn cellulo and in vivo models were employed to investigate the role of SMYD3 in cancer chemoresistance. Analyses of SMYD3-KO cells, drug-resistant cancer cell lines, patients’ residual gastric or rectal tumors that were resected after neoadjuvant therapy and mice models were performed. In addition, the novel SMYD3 covalent inhibitor EM127 was used to evaluate the impact of manipulating SMYD3 activity on the sensitization of cancer cell lines, tumorspheres and cancer murine models to chemotherapeutics (CHTs).ResultsHere we report that SMYD3 mediates cancer cell sensitivity to CHTs. Indeed, cancer cells lacking SMYD3 functions showed increased responsiveness to CHTs, while restoring its expression promoted chemoresistance. Specifically, SMYD3 is essential for the repair of CHT-induced double-strand breaks as it methylates the upstream sensor ATM and allows HR cascade propagation through CHK2 and p53 phosphorylation, thereby promoting cancer cell survival. SMYD3 inhibition with the novel compound EM127 showed a synergistic effect with CHTs in colorectal, gastric, and breast cancer cells, tumorspheres, and preclinical colorectal cancer models.ConclusionsOverall, our results show that targeting SMYD3 may be an effective therapeutic strategy to overcome chemoresistance.

Extracellular vesicles in cancer: Golden goose or trojan horse.

Intercellular communication can be mediated by direct cell-to-cell contact and indirect interactions through secretion of soluble chemokines, cytokines, and growth factors. Extracellular vesicles (EVs) have emerged as important mediators of cell-to-cell and cell-to-environment communications. EVs from tumor cells, immune cells, and stromal cells can remodel the tumor microenvironment and promote cancer cell survival, proliferation, metastasis, immune evasion, and therapeutic resistance. Most importantly, EVs as natural nanoparticles can be manipulated to serve as a potent delivery system for targeted cancer therapy. EVs can be engineered or modified to improve their ability to target tumors and deliver therapeutic substances, such as chemotherapeutic drugs, nucleic acids, and proteins, for the treatment of cancer. This review provides an overview of the biogenesis and recycling of EVs, discusses their roles in cancer development, and highlights their potential as a delivery system for targeted cancer therapy.

Iron promotes ovarian cancer malignancy and advances platinum resistance by enhancing DNA repair via FTH1/FTL/POLQ/RAD51 axis

Iron is crucial for cell DNA synthesis and repair, but an excess of free iron can lead to oxidative stress and subsequent cell death. Although several studies suggest that cancer cells display characteristics of ‘Iron addiction’, an ongoing debate surrounds the question of whether iron can influence the malignant properties of ovarian cancer. In the current study, we initially found iron levels increase during spheroid formation. Furthermore, iron supplementation can promote cancer cell survival, cancer spheroid growth, and migration; vice versa, iron chelators inhibit this process. Notably, iron reduces the sensitivity of ovarian cancer cells to platinum as well. Mechanistically, iron downregulates DNA homologous recombination (HR) inhibitor polymerase theta (POLQ) and relieves its antagonism against the HR repair enzyme RAD51, thereby promoting DNA damage repair to resist chemotherapy-induced damage. Additionally, iron tightly regulated by ferritin (FTH1/FTL) which is indispensable for iron-triggered DNA repair. Finally, we discovered that iron chelators combined with platinum exhibit a synergistic inhibitory effect on ovarian cancer in vitro and in vivo. Our findings affirm the pro-cancer role of iron in ovarian cancer and reveal that iron advances platinum resistance by promoting DNA damage repair through FTH1/FTL/POLQ/RAD51 pathway. Our findings highlight the significance of iron depletion therapy, revealing a promising avenue for advancing ovarian cancer treatment.

Combination of an aurora kinase inhibitor and the ABL tyrosine kinase inhibitor asciminib against ABL inhibitor-resistant CML cells

The development of BCR::ABL1-targeting tyrosine kinase inhibitors (TKIs) has improved the prognosis of patients with chronic myeloid leukemia (CML). However, resistance to ABL TKIs can develop in CML patients due to BCR::ABL1 point mutations and CML leukemia stem cell (LSC). Aurora kinases are essential kinases for cell division and regulate mitosis, especially the process of chromosomal segregation. Aurora kinase members also promote cancer cell survival and proliferation. This study analyzed whether aurora kinases were regulated in the progression of CML. It also evaluated the efficacy of the ABL TKI asciminib and the aurora kinase inhibitor LY3295668. The expressions of AURKA and AURKB were higher in the CML cells compared with normal cells using a public database (GSE100026). Asciminib or LY3295668 alone inhibited CML cells after 72 h, and cellular cytotoxicity was increased. The combined use of Asciminib and LY3295668 increased superior efficacy compared with either drug alone. Colony formation was reduced by cotreatment with asciminib and LY3295668. In the cell-cycle analyses, LY3295668 induced G2/M arrest. Cell populations in the sub-G1 phase were observed when cotreating with asciminib and LY3295668. The combination treatment also changed the mitochondrial membrane potential. In addition, AURKA shRNA transfectant cells had increased asciminib sensitivity. Combining asciminib and aurora kinase inhibition enhanced the efficacy and is proposed as a new therapeutic option for patients with CML. These findings have clinical implications for a potential novel therapeutic strategy for CML patients.

Extra-nuclear TERT counteracts oxidative stress and promotes progression in papillary thyroid carcinoma

The reactivation of TERT is associated with poor outcome in papillary thyroid cancer (PTC). Extra-telomeric functions of TERT were reported, with a protective role against oxidative stress (OS). The aim of the present study was to explore the extra-nuclear TERT localization in PTC and its role in cancer progression.TERT nuclear export under OS were analyzed in K1 PTC cell line. We investigated the role of different TERT localizations using specific TERT constructs that limit its localization to the nucleus or to the mitochondria. The effect of SRC kinase inhibitor PP2, which reduces TERT nuclear export, was investigated as well. Moreover, TERT localization was analyzed in 39 PTC tissues and correlated with the genetic profile and the level of OS, DNA damage and apoptosis in the tumors and with the clinical characteristics of the patients.We demonstrated that TERT is exported from the nucleus in response to OS induced either from H2O2 or the BRAF inhibitor PLX4720. We proved that extra-nuclear TERT reduces mitochondrial OS and induces mitochondrial fragmentation. Moreover, limiting mitochondrial TERT localization reduced proliferation, migration, AKT phosphorylation and glycolysis and increased DNA damage and p21 expression. Finally, in PTC tissues the fraction of mitochondrial/nuclear TERT resulted inversely correlated with OS and p21 expression and associated with tumor persistence.In conclusion, our data indicate that extra-nuclear TERT is involved in reducing the effect of excessive OS, thus promoting cancer cell survival. Extra-nuclear TERT may thus represent a marker of cancer progression and a possible therapeutic target in PTC.

Abstract 4744: Overcoming MK1775 resistance in upper gastrointestinal cancers with combined WEE1 and CHK1 inhibition

Abstract Background: Upper gastrointestinal cancers (UGC), encompassing esophageal and gastric cancers, rank among the top causes of cancer-related deaths globally, with esophageal cancer being the sixth and gastric cancer the fourth leading cause. Esophageal adenocarcinoma (EAC), predominant in the western world, shares histopathological and molecular characteristics with gastric cancer (GC). Despite these similarities, treatment challenges persist, exemplified by the limited efficacy of the WEE1 inhibitor MK1775 in clinical trials due to drug resistance. Our study investigates novel mechanisms underlying this resistance in UGC. Methods and results: RNA sequencing and immunohistochemistry staining demonstrated notable overexpression of WEE1 in over two hundred human EAC and GC samples compared to normal tissues. Concurrently, Western blot analysis revealed a positive correlation between WEE1 and CHK1 overexpression across 16 UGC cell lines. To categorize UGC cells based on their sensitivity to MK1775, we conducted ATP-glo cell viability assays, determining their half-maximal inhibitory concentration (IC50) values. Utilizing nuclear-cytosol separation, our data intriguingly uncovered, for the first time, that in MK1775-resistant cells, MK1775 treatment induces the abnormal translocation of phosphorylated CHK1 (p-CHK1) from the nucleus to the cytoplasm, a shift not observed in MK1775-sensitive cells. Further analysis through Western blot suggested that the cytoplasmic presence of p-CHK1 might trigger the activation of oncogenic STAT3 following MK1775 treatment. This activation appears to stimulate the expression of genes that promote cancer cell survival and drug resistance by enhancing cell survival and inhibiting apoptosis. Notably, combining the WEE1 inhibitor with the CHK1 inhibitor CHIR124 effectively inhibited CHK1 kinase activity and abrogated STAT3 phosphorylation, DNA binding, and transcriptional activity in UGC cells. Our findings strongly indicate that a combined therapy of WEE1 and CHK1 inhibitors could be a promising strategy to overcome MK1775 resistance in UGC cells. Our ongoing research will employ human-derived UGC organoids, patient-derived xenografts (PDXs), and human tissue samples to further validate these findings. Conclusions: Our study underscores the potential of combining WEE1 and CHK1 inhibitors to effectively combat MK1775 resistance in UGC, offering a promising direction for improved treatment strategies and patient outcomes in these challenging malignancies. Citation Format: Krishnapriya Thangaretnam, MD Obaidul Islam, Heng Lu, Dunfa Peng, Nadeem Sidiq Bhat, Mohammed Soutto, EL-Rifai Wael, Zheng Chen. Overcoming MK1775 resistance in upper gastrointestinal cancers with combined WEE1 and CHK1 inhibition [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 4744.

Abstract 581: Identification of potential biomarkers of response to RASMULTI(ON) plus anti-PD-1 combination in preclinical PDAC models

Abstract Mutant KRAS is present in more than 80% pancreatic cancer, promotes cancer cell survival, and protects cancer cells from immune recognition and attack. We have shown that inhibitors of the active state of RAS (RAS(ON)) promote cancer neoantigen recognition and synergize with immunotherapy in preclinical immunogenic models. 1 We evaluated the impact of RMC-6236, an investigational RASMULTI(ON) inhibitor on anti-tumor activity and tumor microenvironment (TME) remodeling in two congenic KRASG12D/+ GEMM-derived PDAC models resistant to anti-PD-1 therapy - KPCY clone 2 (6694c2) and clone 3 (2838c3), developed by Ben Stanger at UPenn. 2 Daily oral administration of RMC-6236 drove tumor regressions in KPCY clone 3, leading to 10% durable complete responses (CRs). In combination with anti-PD-1, 40% of durable CRs were achieved. However, RMC-6236 only drove tumor growth inhibition in KPCY clone 2, and no combination benefit with anti-PD-1. We conducted preclinical biomarker analyses in the TME and periphery to explore differences in response to RMC-6236 alone and in combination with anti-PD-1 in these models. Assessment of baseline predictive biomarkers in tumors elucidated that the KPCY clone 3 cells developed tumors with lower suppressive myeloid cells (gMDSCs) and higher CD8+ T cell infiltration compared to those derived from KPCY clone 2 cells. IHC analyses showed that T cells in clone 3 were in the tumor core, while CD8+ T cells in clone 2 were at the tumor border (the latter frequently observed in PDAC patients). RMC-6236 induced transformation of the TME in favor of anti-tumor immunity in only the KPCY clone 3 - with decreased immunosuppressive gMDSCs, and increased T cells. Limited modulation of the TME was observed in the KPCY clone 2 cells-derived tumors, and gMDSC levels significantly higher than those in baseline tumors of KPCY clone 3. Peripheral immunophenotyping analyses revealed that baseline peripheral suppressive myeloid cells (mMDSCs) were significantly higher in mice harboring tumors originated from KPCY clone 2 cells, suggestive of a more systemic immunosuppressive status. Studies are ongoing to assess additional peripheral immune signatures of preclinical activity of RMC-6236 in combination with anti-PD-1. Taken together, baseline tumor levels of suppressive myeloid cells (gMDSCs) were shown to be a negative predictive biomarker for treatment outcome to RMC-6236 +/- anti-PD-1 in KRAS mutant preclinical PDAC models. Peripheral mMDSCs were shown to be higher in the mice with tumors from the PDAC model not sensitive to the combination of RMC-6236 with anti-PD-1, suggesting that it could be predictive of poor outcome for this combination. These studies in immune-competent models of RAS mutant PDAC allow us to generate tumor and blood-based biomarkers that may predict outcomes to RAS(ON) inhibitor combinations in the clinic. 1 Menard et al. AACR 2023 2 Li et al. Immunity 2018 Citation Format: Lillian Seu, Marie Menard, Jasmine Lee, Christopher Chow, Cristina Blaj, Mariela Moreno Ayala, Nataliya Shifrin, Avery Salmon, Swetha Ganesh, Philip Wig, Alice Kumamoto, Paola Soto-Perez, Tippy Taavili, Vivian Morton, James W. Evans, Jacqueline A. Smith, Elsa Quintana. Identification of potential biomarkers of response to RASMULTI(ON) plus anti-PD-1 combination in preclinical PDAC models [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 581.

Abstract 1690: LSD1 and CoREST2 demethylate STAT3 to promote enteroendocrine cell differentiation in mucinous colorectal cancer

Abstract Across different cancer types, tumor heterogeneity has been shown to drive tumor progression, metastasis, and therapeutic resistance. Adenocarcinoma to neuroendocrine lineage transition is an emergent mechanism of targeted therapy resistance in several cancer types, including lung and prostate cancer. Therefore, understanding the dynamics and mechanisms driving neuroendocrine cell fates in cancer is critical. Mucinous colorectal cancer accounts for upwards of 20% of colorectal cancer cases and is characterized by tumors with mucous accounting for at least 50% of the tumor volume. The normal colon epithelium consists of several specialized cell types, including hormone secreting enteroendocrine cells (EECs), which are the neuroendocrine cell of the intestine. We have previously shown that EEC progenitors are enriched in mucinous colorectal cancer and promote cancer cell survival via secreted factors. Additionally, we have shown that lysine specific demethylase 1 (LSD1) promotes EEC differentiation in these tumors; however, the mechanism by which LSD1 promotes EEC differentiation has remained unknown. Typically to carry out its enzymatic function, LSD1 must be a part of a transcriptional regulatory complex. One such complex is the CoREST complex, which contains LSD1, HDAC1, and one of three CoREST protein family members. Here we report that LSD1 and CoREST2 promote EEC differentiation by demethylating the transcription factor signal transducer and activator of transcription 3 (STAT3) to promote STAT3 chromatin binding. Additionally, we demonstrate that knocking down CoREST2 decreases tumor growth and lung metastases of mucinous colorectal cancer cells injected orthotopically into the colons of immunocompromised mice. Furthermore, we utilized single cell multi-omics that combines single cell RNA sequencing with single cell ATAC sequencing to show that during EEC differentiation there is an increase in chromatin accessibility at regulatory regions of known EEC-promoting transcription factors. Finally, through our single cell multi-omics analysis we identify a new rare cell type with neuron-like but not secretory characteristics. Mucinous colorectal cancer is an aggressive and chemotherapeutically intractable form of CRC that is enriched for EEC progenitors. Our data demonstrates that LSD1 and STAT3 promote EEC differentiation, suggesting that inhibiting LSD1 and or STAT3 may have benefits for patients with mucinous colorectal cancer. Citation Format: Christopher A. Ladaika, Ahmed H. Ghobashi, William C. Boulton, Heather M. O'Hagan. LSD1 and CoREST2 demethylate STAT3 to promote enteroendocrine cell differentiation in mucinous colorectal 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 1690.

Abstract 3256: Novel SOX2-targeting bioPROTACs for the treatment of cancers

Abstract Targeted protein degradation (TPD) technology has been developed showing the therapeutic potential to modulate historically ‘undruggable’ disease-associated proteins. Currently, proteolysis targeting chimeras (PROTACs) using small-molecule binders represent the TPD field, and PROTAC-induced proximity between target proteins and E3 ligases enables specific degradation of target proteins. However, substantial amounts of clinically relevant cancer targets such as transcription factors lack active sites or allosteric binding pockets for small molecules which challenges the development of classical PROTACs for these targets. SOX2 is one of the pluripotency transcription factors and has been well-known for its crucial role in tumor development through promoting cancer cell survival, metastasis, stemness, and drug resistance. Numerous studies have also reported SOX2 amplification and overexpression in various tumor tissues. Thus, SOX2 has been validated as an attractive anticancer target, but directly targeting SOX2 has been proved to be difficult since SOX2 is an “undruggable” transcription factor. EPD Biotherapeutics develops EPDegTM, a next-generation engineered protein degrader platform in order to overcome the limitations of small molecule-based PROTAC. Here we report a series of SOX2-targeting bioPROTACs, being composed of a nanobody against SOX2 with high affinity and several ubiquitin E3 ligases with deletion of natural substrate binding domain to allow highly selective degradation of SOX2 protein. To evaluate the in vitro efficacy, SOX2 bioPROTACs were generated by mRNA IVT with CleanCap ® and modified uridine. First, robust degradation of SOX2 with a half-maximal degradation concentration (DC50) of the picomolar range was observed with the treatment of SOX2 bioPROTACs in multiple SOX2-overexpressing cancer cell lines. The degradation was ubiquitin- and proteosome-dependent manner, and neither nanobody nor ubiquitin E3 ligase alone affected SOX2 protein level. Then, SOX2 bioPROTACs led to significant cancer cell growth inhibition caused by apoptotic cell death with a half-maximal growth inhibition concentration (GI50) of the picomolar range. Notably, chemo-resistant cancer cell lines expressing SOX2 protein in high levels were sensitized to chemotherapy in combination with our SOX2 bioPROTACs. Furthermore, i.v. injection of one of our lead candidates formulated with LNP significantly inhibited tumor growth in the A549 xenograft model, proving the in vivo anti-tumor efficacy of SOX2 degraders for the first time in the world. Taken together, EPD’s SOX2 bioPROTACs show potent in vitro and in vivo anti-tumor efficacy as first-in-class SOX2 degraders and provide new insights for the therapeutic potential of transcription factor-degrading strategies. Citation Format: Jaehyun Choi, Saeyi Lim, Xuelian Bai, Hyerin Song, Yeonji Oh, Yeryoung Yong, Seunghyun Lee. Novel SOX2-targeting bioPROTACs for the treatment of cancers [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 3256.

Abstract 3082: The liver microenvironment promotes glycolysis in metastatic colorectal cancer by activating her3

Abstract Background: Liver metastasis occurs in ~80% of all metastatic colorectal cancer (mCRC) and the liver has a unique endothelial cell (EC)-rich microenvironment that promotes cancer cell survival. We previously reported that liver ECs secrete soluble factors to promote CRC growth via activating CRC-associated the HER3 signaling pathway. The present study reports that ECs promote mCRC growth by shifting cancer metabolism towards glycolysis and elucidates the involved signaling pathway. Methods: We performed mass spectrometry to profile the metabolic changes in mCRC treated with/without HER3 inhibition in a syngeneic, orthotopic mCRC model. We also performed Seahorse and lactate ELISA assays to determine the effect of liver EC-secreted factors on CRC metabolism. To further determine the role of HER3 in promoting glycolysis and CRC growth in the liver, we used siRNA silencing of HER3 and pharmacological inhibition of HER3 downstream signaling proteins to identify the key mediator(s) of HER3-induced metabolic shift in CRC. Results: We determined that HER3 inhibition leads to increased oxidative phosphorylation metabolites (OXPHOS) and decreased glycolysis metabolites in CRC liver metastases. Conditioned medium containing EC-secreted factors decreased oxygen consumption (determined by Seahorse, a readout of OXPHOS) and increased lactate secretion (determined by ELISA, a readout of glycolysis) in CRC. As HER3 inhibition reversed the metabolic shift and increased OXPHOS as an alternative survival strategy, we found that simultaneous inhibition of HER3 and OXPHOS synergistically blocked CRC cell and tumor growth. Lastly, we found that PFK2 is activated by HER3 signaling, determined by phosphorylation, and determined that the HER3-AKT-PFK2 signaling axis is the key mediator of EC-induced metabolic shifts. Conclusions: We identified that the liver microenvironment activates HER3 and the downstream AKT-PFK2 signaling to promote glycolysis and growth in CRC liver metastases by activating AKT-PFK2-dependent glycolysis. Our findings highlighted the potential of combining HER3 and OXPHOS inhibitions as a potential strategy for treating patients with mCRC. Citation Format: Moeez Ghani Rathore, Kimberly Curry, Christina Boutros, Zhenghe Wang, Jordan Winter, Rui Wang. The liver microenvironment promotes glycolysis in metastatic colorectal cancer by activating her3 [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 3082.

Abstract 4616: TBK1 and GSK3 mediated TRAF2 phosphorylation confers resistance to PI3K-AKT inhibition in breast cancer cells

Abstract Breast cancer (BC) is the most frequently diagnosed malignancy and the second leading cause of cancer mortality in Western women. As is the case for most other solid tumors, metastasis and drug resistance are the main causes of death. In ~80% of BC cases, the PI3K-AKT pathway is aberrantly activated, due to the alterations in genes encoding the pathway components, such as Ras, Her2, PTEN, PIK3C and AKT. Consequently, over 100 clinical trials are currently underway worldwide to evaluate the therapeutic efficacy of PI3K and AKT inhibitors in BC; however, initial data revealed that inhibition of this pathway is either not effective or often results in development of resistance and relapse of the disease. Thus, identification of additional targets and therapeutic combinations are urgently needed. We previously reported that TBK1-mediated TRAF2 Ser-11 phosphorylation enhances NF-κB activation to promote cancer cell survival under conditions of cellular stresses. Recently, we discovered that GSK3β directly phosphorylates TRAF2 at Thr-7 upon Ser-11 phosphorylation by TBK1, and that phosphorylation of both Thr-7 and Ser-11 is essential for sustained NF-κB activation and BC cell resistance to PI3K-AKT inhibition. TBK1 and its close homologue IKKε are overexpressed in ~70% of BC and play critical roles in BC cell survival. GSK3β is a constitutively active kinase activity in unstimulated normal cells, but its activity is suppressed in most cancer cell types by AKT-mediated phosphorylation. These published reports and our new findings suggest that inhibition of AKT in BC cells leads to increased phosphorylation of TRAF2 by TBK1 and GSK3β, which confers resistance to PI3K-ATK inhibition. Bioinformatic analyses revealed that TRAF2 and GSK3β are also overexpressed in invasive BC, and significantly correlate with poor prognosis. As expected, inhibition of both AKT and TBK1 or GSK3b synergistically induced apoptosis in BC cell lines in vitro and significantly suppressed xenograft BC tumor growth in vivo. Thus, our data suggest that further investigation of the efficacies of combined PI3K/AKT and TBK1/GSK3β inhibition in PDX models of BC has the potential to develop new strategies for BC treatment. Citation Format: Hasem Habelhah, Laiqun Zhang. TBK1 and GSK3 mediated TRAF2 phosphorylation confers resistance to PI3K-AKT inhibition in breast 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 4616.

Abstract 618: Preclinical assessment of APL-030, a selective and orally bioavailable inhibitor of the integrated stress response regulator GCN2 with activity against acute myeloid leukemia

Abstract GCN2 (EIF2AK4) is an evolutionarily conserved kinase and a pivotal regulator of the Integrated Stress Response (ISR), an adaptive cellular program triggered primarily by amino acid scarcity. Active GCN2 phosphorylates the translation initiation factor eIF2α, resulting in the attenuation of global protein synthesis and a largely ATF4-driven program of resource enhancement. GCN2 promotes cancer cell survival under conditions of microenvironmental or drug-induced intracellular nutrient scarcity. Here, we describe the preclinical development of APL-030, a novel and selective ATP-competitive inhibitor of GCN2. APL-030 was confirmed as a potent inhibitor of GCN2 in an on-target biochemical kinase assay with a Ki of 4.4nM. In a cell-based assay APL-030 decreased eIF2α phosphorylation in a dose-dependent manner via direct GCN2 inhibition with an IC50 of 50.8nM. In acute myeloid leukemia (AML) cell lines GCN2 inhibition resulted in a decrease in gene expression of effector genes CHAC1 and DDIT3. Moreover, treatment with APL-030 dose-dependently decreased cell viability in multiple AML cell lines, which was accompanied by an increase in caspase 3/7 activity. Bulk mRNA sequencing of MOLM-13 AML cells showed that APL-030 inhibited the extensive transcriptome changes triggered by glutamine depletion, in line with a high degree of selectivity of APL-030 for GCN2-mediated ISR signaling. In cells grown in rich medium, APL-030 triggered a largely ISR-independent transcriptome response that was dominated by the enrichment of pathways related to cell cycle as well as RNA and protein metabolism. In vivo, APL-030 treatment of an AML xenograft animal model completely inhibited tumor growth and prolonged survival time. Inhibition of AML tumor growth was shown to be a consequence of decreased cell viability and induction of caspase-dependent apoptosis. APL-030 was also tested on primary diagnostic AML cells, which were assessed for cell death by flow cytometry following staining for Annexin-V, 7-AAD, CD45, CD34, and CD38. Treatment with APL-030 caused a statistically significant increase in AML cell death in all six samples studied. Three samples exhibited a measurable CD34+/CD38- compartment, enabling evaluation of this leukemic stem cell-enriched population. APL-030 caused a statistically significant increase in cell death in the leukemic stem cell-enriched population in all three samples studied. APL-030 is a novel GCN2 inhibitor that that has shown encouraging efficacy in preclinical studies using both AML cell lines and patient-derived AML samples. Based on these preclinical results, a phase 1/2 study is planned in hematological tumors. Citation Format: Monica Roman-Trufero, Gavin Whitlock, Matthew Fuchter, Maxmila Jeyakumar, Panagiota Chaida, Sereina Annik Herzog, Armin Zebisch, Richard Butt, Holger W. Auner, Nadine Clemo. Preclinical assessment of APL-030, a selective and orally bioavailable inhibitor of the integrated stress response regulator GCN2 with activity against acute 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 618.

Abstract 4451: Regulation of lipid metabolism and ferroptosis by DAXX

Abstract Intracellular lipid production in cancer cells supplies lipids to synthesize cell membranes and signaling molecules during rapid cell proliferation and tumor growth. Cancer cells also utilize fatty acid oxidation (FAO) to generate ATP to meet their energy demand. Notably, lipid metabolites can inhibit and trigger ferroptosis due to iron-dependent oxidation of polyunsaturated fatty acids (PUFAs). Therefore, identifying regulators that maintain the intricate balance of lipid biosynthesis required for cell proliferation and survival is critical in cancer biology and therapy. Lipid metabolism is regulated by two oncogenic signaling pathways: the RAS-RAF-MEK-MAPK and the mammalian target of rapamycin (mTOR) pathways. About 30% of all cancers harbor constitutively active mutations in KRAS, HRAS, or NRAS, resulting in hyperactive RAS-RAF-MEK-MAPK signaling to drive tumorigenesis, metastatic progression, immune evasion, and resistance to therapy. KRAS regulates lipid uptake, lipid synthesis, and FAO. mTOR is a serine/threonine kinase acting as a key intracellular signaling hub to regulate nutrient homeostasis, metabolism, protein synthesis, and autophagy. The mTORC1 complex promotes lipogenesis. The RAS and mTOR signaling pathways exhibit both positive and negative cross-regulation. The coordinated activity of both pathways is critical to sustained tumor growth. Notably, mTORC1 signaling inhibition enhances RAS-RAF-MEK-MAPK signaling to promote cancer cell survival and proliferation. Furthermore, constitutive mTORC1 signaling induces cell death when the supply of unsaturated FAs is limited. However, the molecular link for coordinating the activity of the RAS and mTOR signaling pathways remains poorly defined. Death domain-associated protein (DAXX) is essential for mouse embryonic development and has ill-defined pro-cell survival functions. Our lab has a long-standing interest in deciphering DAXX’s complex biological functions. Our recent study shows that DAXX drives tumorigenesis by promoting lipogenic gene expression and lipid synthesis through interacting with sterol regulatory element-binding proteins SREBP1 and SREBP2 (SREBP1/2) (Mahmud et al., 2023, PMID 37045819). Unexpectedly, our new data reveal that DAXX inhibits ferroptosis. Significantly, phosphorylation via RAS signaling appears to regulate DAXX’s activity in lipid synthesis and resistance to ferroptosis. Remarkably, disruption of DAXX-mediated lipid synthesis potentiates ferroptosis due to mTORC1 activity. Based on these observations, we propose that stimulated by the RAS-RAF-MEK-MAPK oncogenic signaling, DAXX promotes tumorigenesis through upregulating genes for lipid synthesis and resistance to ferroptosis, and that the lack of DAXX-mediated lipid synthesis leads to sensitization of cells to ferroptosis due to mTORC1’s proliferative effects. (Supported by FDOH grant 23K03). Citation Format: Wenlin Yang, Nikee Awasthee, Qi Chen, Seth Hale, Daiqing Liao. Regulation of lipid metabolism and ferroptosis by DAXX [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 4451.

METTL16 deficiency attenuates apoptosis through translational control of extrinsic death receptor during nutrient deprivation

METTL16 is a well-characterized m6A methyltransferase that has been reported to contribute to tumorigenesis in various types of cancer. However, the effect of METTL16 on tumor progression under restricted nutrient conditions, which commonly occur in tumor microenvironment, has yet to be elucidated. Herein, our study initially reported the inhibitory effect of METTL16 depletion on apoptosis under amino acid starvation conditions. Mechanistically, we determined that the METTL16 knockdown represses the expression of extrinsic death receptors at both transcription and translation levels. Depletion of METTL16 prevented protein synthesis of GCN2, resulting in diminished ATF4 expression in a GCN2-eIF2α-dependent manner. Reduction of ATF4 further declined the expression of apoptotic receptor protein DR5. Meanwhile, METTL16 deficiency directly hampered protein synthesis of FADD and DR5, thereby impairing apoptosis and promoting cancer cell survival. Taken together, our study provides novel evidence for the involvement of METTL16 in regulating cancer progression, suggesting that METTL16 as a potential therapeutic target for cancer treatment.

MDM2 provides TOP2 poison resistance by promoting proteolysis of TOP2βcc in a p53-independent manner

DNA topoisomerase II (TOP2) is an enzyme that performs a critical function in manipulating DNA topology during replication, transcription, and chromosomal compaction by forming a vital intermediate known as the TOP2-DNA cleavage complex (TOP2cc). Although the TOP2cc is often transient, stabilization can be achieved by TOP2 poisons, a family of anti-cancer chemotherapeutic agents targeting TOP2, such as etoposide (VP-16), and then induce double-strand breaks (DSBs) in cellular DNA. TOP2cc first needs to be proteolyzed before it can be processed by TDP2 for the removal of these protein adducts and to produce clean DNA ends necessary for proper repair. However, the mechanism by which TOP2βcc is proteolyzed has not been thoroughly studied. In this study, we report that after exposure to VP-16, MDM2, a RING-type E3 ubiquitin ligase, attaches to TOP2β and initiates polyubiquitination and proteasomal degradation. Mechanistically, during exposure to VP-16, TOP2β binds to DNA to form TOP2βcc, which promotes MDM2 binding and subsequent TOP2β ubiquitination and degradation, and results in a decrease in TOP2βcc levels. Biologically, MDM2 inactivation abrogates TOP2β degradation, stabilizes TOP2βcc, and subsequently increases the number of TOP2β-concealed DSBs, resulting in the rapid death of cancer cells via the apoptotic process. Furthermore, we demonstrate the combination activity of VP-16 and RG7112, an MDM2 inhibitor, in the xenograft tumor model and in situ lung cancer mouse model. Taken together, the results of our research reveal an underlying mechanism by which MDM2 promotes cancer cell survival in the presence of TOP2 poisons by activating proteolysis of TOP2βcc in a p53-independent manner, and provides a rationale for the combination of MDM2 inhibitors with TOP2 poisons for cancer therapy.

KH-like Domains in PARP9/DTX3L and PARP14 Coordinate Protein–Protein Interactions to Promote Cancer Cell Survival

Certain members of the ADP-ribosyltransferase superfamily (ARTD or PARP enzymes) catalyse ADP-ribosylation in response to cellular stress, DNA damage and viral infection and are upregulated in various tumours. PARP9, its binding partner DTX3L and PARP14 protein levels are significantly correlated in head and neck squamous cell carcinoma (HNSCC) and other tumour types though a mechanism where PARP9/DTX3L regulates PARP14 post-transcriptionally. Depleting PARP9, DTX3L or PARP14 expression in HNSCC or HeLa cell lines decreases cell survival through a reduction of proliferation and an increase in apoptosis. A partial rescue of survival was achieved by expressing a PARP14 truncation containing a predicted eukaryotic type I KH domain. KH-like domains were also found in PARP9 and in DTX3L and contributed to protein–protein interactions between PARP9-DTX3L and PARP14-DTX3L. Homodimerization of DTX3L was also coordinated by a KH-like domain and was disrupted by site-specific mutation. Although, cell survival promoted by PARP14 did not require ADP-ribosyltransferase activity, interaction of DTX3L in vitro suppressed PARP14 auto-ADP-ribosylation and promoted trans-ADP-ribosylation of PARP9 and DTX3L. In summary, we characterised PARP9-DTX3L-PARP14 interactions important to pro-survival signalling in HNSCC cells, albeit in PARP14 catalytically independent fashion.

5-Oxo-ETE/OXER1: A Link between Tumor Cells and Macrophages Leading to Regulation of Migration.

Chronic inflammation is an important factor in the development of cancer. Macrophages found in tumors, known as tumor associated macrophages (TAMs), are key players in this process, promoting tumor growth through humoral and cellular mechanisms. 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), an arachidonic acid metabolite, has been described to possess a potent chemoattractant activity for human white blood cells (WBCs). The biological actions of 5-oxo-ETE are mediated through the GPCR 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid receptor (OXER1). In addition, we have previously reported OXER1 as one of the membrane androgen receptors with testosterone antagonizing 5-oxo-ETE's actions. OXER1 is highly expressed in inflammatory cells and many normal and cancer tissues and cells, including prostate and breast cancer, promoting cancer cell survival. In the present study we investigate the expression and role of OXER1 in WBCs, THP-1 monocytes, and THP-1 derived macrophages, as well as its possible role in the interaction between macrophages and cancer cells (DU-145 and T47D). We report that OXER1 is differentially expressed between WBCs and macrophages and that receptor expression is modified by LPS treatment. Our results show that testosterone and 5-oxo-ETE can act in an antagonistic way affecting Ca2+ movements, migration, and cytokines' expression in immune-related cells, in a differentiation-dependent manner. Finally, we report that 5-oxo-ETE, through OXER1, can attract macrophages to the tumor site while tumor cells' OXER1 activation in DU-145 prostate and T47D breast cancer cells, by macrophages, induces actin cytoskeletal changes and increases their migration.

Efficacy of ABCA1 Transporter Proteins in Patients with Endometrial Cancer: an In Vitro Study.

Endometrial carcinoma (EC) is a typical gynecological malignant tumor that occurs more frequently every year. Obesity is a significant contributor to the development of EC and its prognosis. Lipid metabolism and malignant tumors have a long history of association. Elevated cholesterol levels are made possible by adenosine triphosphate-binding cassette protein A1 (ABCA1) deficiency, which eventually promotes cancer cell survival. The aim of this study was to examine at the ABCA1 gene expression levels in EC patients. The relationship between ABCA1 and the occurrence, progression, and prognosis of EC is discussed in this article as a potential mechanism. The samples of 45 endometrial adenocarcinoma patients were retrospectively included in this study and they were further divided into Grade 1 (15), Grade 2 (15), Grade 3 (15) tumors, control group. Twenty-nine endometrial tissues without a confirmed diagnosis of endometrial cancer made up the control group. ABCA1 gene expression was examined using real-time polymerase chain reaction. According to the results, the gene expressions of the patient group were higher than the control group When each Grade was compared with the control group, statistically significant results were obtained. After analyzing the data, it was found that the patient group was generally higher than the control group (p < 0.05) and there were differences in the grades of the patient group (p < 0.05). When the ABCA1 expressions of the grade groups and control groups were compared separately, a difference was found between Grade 1, Grade 2 and Grade 3 and the control group (p= 0.0001). According to the findings of our study, a key component in the growth of EC tumors is the increase in cholesterol production caused by a reduction in ABCA1.

Revealing profile of cancer-educated platelets and their factors to foster immunotherapy development

Among multiple hemostasis components, platelets hyperactivity plays major roles in cancer progression by providing surface and internal components for intercellular crosstalk as well as by behaving like immune cells. Since platelets participate and regulate immunity in homeostatic and disease states, we assumed that revealing platelets profile might help in conceiving novel anti-cancer immune-based strategies. The goal of this review is to compile and discuss the most recent reports on the nature of cancer-associated platelets and their interference with immunotherapy. An increasing number of studies have emphasized active communication between cancer cells and platelets, with platelets promoting cancer cell survival, growth, and metastasis. The anti-cancer potential of platelet-directed therapy has been intensively investigated, and anti-platelet agents may prevent cancer progression and improve the survival of cancer patients. Platelets can (i) reduce antitumor activity; (ii) support immunoregulatory cells and factors generation; (iii) underpin metastasis and, (iv) interfere with immunotherapy by expressing ligands of immune checkpoint receptors. Mediators produced by tumor cell-induced platelet activation support vein thrombosis, constrain anti-tumor T- and natural killer cell response, while contributing to extravasation of tumor cells, metastatic potential, and neovascularization within the tumor. Recent studies showed that attenuation of immunothrombosis, modulation of platelets and their factors have a good perspective in immunotherapy optimization. Particularly, blockade of intra-tumoral platelet-associated programmed death-ligand 1 might promote anti-tumor T cell-induced cytotoxicity. Collectively, these findings suggest that platelets might represent the source of relevant cancer staging biomarkers, as well as promising targets and carriers in immunotherapeutic approaches for combating cancer.