Abstract Hypoxia-inducible factor 1-alpha (HIF-1α) plays a pivotal role in orchestrating cellular responses to hypoxia, influencing cancer cell survival and progression. Our previous work identified a non-canonical mechanism wherein Smurf2 mediates HIF-1α degradation under CDK4/6 inhibition. Through proteomic analysis, we discovered that serine 451 phosphorylation occurs on HIF-1α but not in palbociclib-treated samples. Point mutations at this site, substituting serine with alanine, resulted in decreased HIF-1α levels and enhanced interaction with Smurf2. Intriguingly, under palbociclib treatment, we observed phosphorylation at the serine 643 site. This site has been previously associated with MAPK-dependent regulation of HIF-1α localization and activity (Ilias Mylonis, et al., 2006), particularly relevant given the reported MAPK reliance in acquired CDK4/6 inhibitor-resistant scenarios (Renée de Leeuw, et al., 2018). To explore therapeutic implications, we investigated the impact of combined CDK4/6 (palbociclib) and MEK1/2 inhibition (trametinib, selumetinib, PD98059, U0126). Dual inhibition robustly reduced HIF-1α expression in colorectal cancer cells (HCT116, SW480) and suppressed HIF-1α activity in luciferase reporter assays. This effect extended to synergistic inhibition of cell viability under both normoxia and hypoxia in HCT116 and SW480 cells. Such effect is also applicable to other cancer types and cell lines (e.g. U251). In summary, our findings unveil a phosphorylation site on HIF-1α associated with CDK4/6 activity, influencing its protein stabilization. This discovery supports the rationale for combining CDK4/6 and MEK1/2 inhibition as a promising strategy in the treatment of solid tumors. Citation Format: Shuai Zhao, Lanlan Zhou, Shengliang Zhang, Wafik S. El-Deiry. Targeting the convergence on HIF-1α of CDK4/6 and MAPK pathway: Implications for enhanced anticancer strategies [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 376.

Apoptosis is a form of programmed cell death that is mediated by intrinsic and extrinsic pathways. Dysregulation of and resistance to cell death are hallmarks of cancer. For over three decades, the development of therapies to promote treatment of cancer by inducing various cell death modalities, including apoptosis, has been a main goal of clinical oncology. Apoptosis pathways also interact with other signaling mechanisms, such as the p53 signaling pathway and the integrated stress response (ISR) pathway. In addition to agents directly targeting the intrinsic and extrinsic pathway components, anticancer drugs that target the p53 and ISR signaling pathways are actively being developed. In this Review, we discuss selected and promising anticancer therapies in various stages of development, including drug targets, mechanisms, and resistance to related treatments, focusing especially on B cell lymphoma 2 (BCL-2) inhibitors, TRAIL analogues, DR5 antibodies, and strategies that target p53, mutant p53, and the ISR.

Single-agent TAS102 (trifluridine/tipiracil) and regorafenib are FDA-approved treatments for metastatic colorectal cancer (mCRC). We previously reported that regorafenib combined with a fluoropyrimidine can delay disease progression in clinical case reports of multidrug-resistant mCRC patients. We hypothesized that the combination of TAS102 and regorafenib may be active in CRC and other gastrointestinal (GI) cancers and may in the future provide a treatment option for patients with advanced GI cancer. We investigated the therapeutic effect of TAS102 in combination with regorafenib in preclinical studies employing cell culture, colonosphere assays that enrich for cancer stem cells, and in vivo. TAS102 in combination with regorafenib has synergistic activity against multiple GI cancers in vitro including colorectal and gastric cancer, but not liver cancer cells. TAS102 inhibits colonosphere formation and this effect is potentiated by regorafenib. In vivo anti-tumor effects of TAS102 plus regorafenib appear to be due to anti-proliferative effects, necrosis and angiogenesis inhibition. Growth inhibition by TAS102 plus regorafenib occurs in xenografted tumors regardless of p53, KRAS or BRAF mutations, although more potent tumor suppression was observed with wild-type p53. Regorafenib significantly inhibits TAS102-induced angiogenesis and microvessel density in xenografted tumors, as well inhibits TAS102-induced ERK1/2 activation regardless of RAS or BRAF status in vivo. TAS102 plus regorafenib is a synergistic drug combination in preclinical models of GI cancer, with regorafenib suppressing TAS102-induced increase in microvessel density and p-ERK as contributing mechanisms. The TAS102 plus regorafenib drug combination may be further tested in gastric and other GI cancers.

TPS3167 Background: Cyclin-dependent kinases 4/6 (CDK4/6) promote cell-cycle progression and cancer growth. CDK4/6 inhibitors (CDK4/6i), in combination with hormonal therapy, increase progression-free and overall survival (PFS and OS) in breast cancer but resistance to CDK4/6i is associated with genomic abnormalities including Rb-deficiency. Combined CDK4/6i/HSP90i has been shown to inhibit HIF1-alpha including in tumor cells with Rb-deficiency. Synergy between multiple CDK4/6i’s and HSP90i’s has suggested a drug class effect for combination. In CDK4/6i-treated cells, E3-ubiquitin ligase Smurf2 associates with and targets degradation of HIF1-alpha independent of VHL or hypoxia. BrUOG 387 investigates whether dual targeting of CDK4/6 and HSP90, through HIF1-alpha in patients with ER/PR+ Her2- advanced breast cancer progressing on CDK4/6i and patients with refractory Rb-deficient tumors is safe and reasonably tolerated. Methods: A phase Ib open-label single-arm dose de-escalation study is evaluating safety, tolerability as primary outcomes, recommended doses for cohort expansion and preliminary efficacy of CDK4/6i and HSP90i (IND163592; NCT05655598; the only HSP90i combination study in US). Patients with ER/PR+ Her2- advanced breast cancer progressing on CDK4/6i, and in cohort expansion, patients with Rb-deficient solid tumors (SCLC, soft tissue sarcoma, endometrial and bladder cancer) are included. CDK4/6i (palbociclib at dose previously tolerated up to 125 mg daily PO D1-21 of 28 day cycle) in combination with HSP90i (TAS-116/pimitespib starting at 120 mg 5 days on 2 days off, for D1-28 of cycle) is used in 3+3 dose-de-escalation design. The study includes PK analysis, and PD analysis will include HIF1/2, Smurf2, Rb, HIF targets VEGF, erythropoietin, Glut1, proliferation, cell death, cancer stem cells, NK, T-cells, RNA-seq and angiogenesis in pre- and post-treatment biopsies. Biostatistical design and analysis includes 3 dose levels of TAS-116 + palbociclib (level 0: TAS-116 120 mg, palbociclib 125 mg; level -1: TAS-116 80 mg, palbociclib 125 mg); level -2: TAS-116 40 mg, palbociclib 125 mg). Limiting Toxicities will include grade 4 neutropenia lasting > 7 days, neutropenic fever, grade 4 thrombocytopenia or Grade 3 non-hematologic toxicity not controlled with medical management. The study will enroll a total of approximately 15-27 patients (6-18 patients in dose de-escalation portion of the study and 9 additional patients in the cohort expansion phase). Secondary outcome analyses include response rate (CR, PR) measured by RECIST v.1.1 with exact two-sided 95% confidence intervals. OS, PFS, TTP, and DOR will be summarized using methods of Kaplan and Meier. Clinical trial information: NCT05655598 .

Abstract Hypoxia-inducible factors (HIFs) act as transcription factors and play an essential role in cellular and systemic responses to low oxygen environments. HIF-1α expression is induced in acute hypoxia typically through failure of its ubiquitination and degradation mediated by Von Hippel-Lindau (VHL). Previously we discovered a non-canonical mechanism where the SMAD specific E3 ubiquitin protein ligase 2 (Smurf2) promotes the ubiquitination of HIF-1α and reduces HIF-1α level in HCT116 colorectal cancer cells. Smurf2 is a HECT-type ubiquitin ligase known to interact with Smad proteins, leading to their ubiquitination and proteasomal degradation. Overexpression of Smurf2 decreased HIF-1α expression in HCT116 and SW480 colorectal cancer cells under hypoxia as well as in RCC4 VHL-deficient kidney renal clear cell carcinoma cells under normoxia. Treatment with MG132 at least partially rescued the expression of HIF-1α following Smurf2 overexpression, indicating involvement of proteasome-dependent degradation in Smurf2-mediated HIF-1α destabilization. Knockdown of SMURF2 increased HIF-1α expression under normoxia in HCT116 and RCC4 cells. To investigate the effect of Smurf2 inhibition, we tested a selective reversible inhibitor of HECT E3 ubiquitin ligases, heclin, along with its analogues, PYR-41, C646 and 4E1RCat. Treatment with heclin increased HIF-1α expression in HCT116 under hypoxia as well as in SW480 and RCC4 cells under normoxia in a dose-dependent manner. PYR-41 elevated the level of HIF-1α level in HCT116 cells under hypoxia, in RCC4 cells under normoxia and in SW480 cells under both normoxia and hypoxia. To examine the effect on HIF transcriptional activity, we performed luciferase reporter assay using plasmids containing the hypoxia response element (HRE) from the PGK1 promoter and the VEGF promoter, respectively. PYR-41 induced transcriptional activity on PGK1-HRE in both HCT116 and SW480 cells. 4E1RCat robustly activated transcription on both VEGF-HRE and PGK1-HRE in HCT116 and SW480 cells. In summary, Smurf2 targets HIF-1α for ubiquitination and degradation independently of oxygen concentration and inhibition of Smurf2 to stimulate HIF activity under normoxia or hypoxia may be beneficial in pathological circumstances featuring anemia and hypoxemia. Citation Format: Shuai Zhao, Wafik S. El-Deiry. Inhibition of Smurf2 E3 ubiquitin ligase by heclin and its analogues enhances HIF-1α expression and transcriptional activity in normoxia or hypoxia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4812.

Glioblastoma (GBM) is the most common malignant brain and other central nervous system (CNS) tumor, which comprises 47.7% of all cases. The standard treatment options are surgery, radiation therapy, and chemotherapy using temozolomide. GBM is fast-growing, aggressive, and results in poor overall survival, approximately 40% in the first year and 17% in the second year. Hypoxia, the lack of sufficient oxygen in tissues, is the hallmark of GBM and can induce drug resistance and inhibit anti-tumor immune responses. Previous studies show that CDK inhibitors can destabilize HIF1, a key regulator of hypoxia induced apoptosis and p53 stabilization. Palbociclib, a potent oral inhibitor of CD4/6, is undergoing clinical trials for GBM treatment. However, it was not an effective treatment for recurrent GBM when used as alone in a Phase II study. Thus, we are exploring the cytotoxicity of palbociclib in combination with an anti-PD1 drug pembrolizumab, which is proven to be highly effective in other cancer types. Preliminary studies use CellTitlerGLO viability assay to determine the IC50s for Palbociclib at 24, 48, and 72 hours in both normoxia and hypoxia conditions. Immune cell co-culture, comprising of GBM cell lines plus TALL104 human leukemia T cells, was used to determine the amount of tumor cell death with palbociclib, pembrolizumab and a combination at different time points in both normoxia and hypoxia conditions. Our results are providing insights into improving immune checkpoint therapy for GBM patients. Citation Format: Yutong Xia, Lanlan Zhou, Shuai Zhao, Wafik S. El-Deiry. Combination of palbociclib, a potent CDK4/6 inhibitor, with anti-PD1 drug pembrolizumab treatment to promote T-cell mediated glioblastoma tumor cell death under hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3254.

TAS-102 is an oral formulation consisting of trifluridine (FTD) and thymidine phosphorylase inhibitor tipiracil hydrochloride (TPI). Regorafenib is a multi-targeted tyrosine kinase inhibitor, it inhibits the activities of VEGFR2 and 3, Ret, Kit, PDGFR and Raf kinases and results in the inhibition of tumor angiogenesis and cell proliferation. TAS102 or regorafenib monotherapy can lead to an overall survival benefit in metastatic colorectal cancer (mCRC) patient previously treated with conventional chemotherapy and target therapy (Arnold et al., 2018). TAS102 in combination with bevacizumab, a monoclonal antibody against vascular endothelial growth factor A (VEGF-A), is associated with a significant and clinically relevant improvement in progression-free survival in colorectal cancer (C-TASK FORCE)(Kuboki et al., 2017; Pfeiffer et al., 2020). FTD can synergize with nintedanib to inhibit the growth of colorectal cancer xenografts(Suzuki, Nakagawa, Matsuoka, & Takechi, 2016). We previously reported that regorafenib combined with a fluoropyrimidine can delay disease progression in case reports of multidrug-resistant mCRC patients(Marks et al., 2015). Therefore, we hypothesized that the combination of TAS102 and regorafenib may be active in CRC and other gastrointestinal (GI) cancers, and may in the future provide a treatment option for patients with advanced GI cancer. We investigated the therapeutic effect of TAS102 in combination with regorafenib in preclinical studies employing cell culture, colonosphere assays that enrich for cancer stem cells, and in vivo. We found that TAS102 in combination with regorafenib has synergistic activity against multiple GI cancers in vitro including colorectal and gastric cancer, but not liver cancer cells. TAS102 inhibits colonosphere formation and this effect is potentiated by regorafenib. In vivo anti-tumor effects of TAS102 plus regorafenib appear to be due to anti-proliferative effects, necrosis and angiogenesis inhibition. Growth inhibition by TAS102 plus regorafenib occurs in xenografted tumors regardless of p53, KRAS or BRAF mutations, although more potent tumor suppression was observed with wild-type p53. Regorafenib significantly inhibits TAS102-induced angiogenesis and microvessel density in xenografted tumors, and inhibits TAS102-induced ERK1/2 activation regardless of RAS or BRAF status in vivo. Collectively, TAS102 plus regorafenib is a synergistic drug combination in preclinical models of GI cancer, with regorafenib suppressing TAS102-induced increase in microvessel density and p-ERK as contributing mechanisms. The drug combination may be further tested in gastric and other GI cancers. Citation Format: Jun Zhang, Lanlan Zhou, Shuai Zhao, Wafik S. El-Deiry. TAS102 synergizes with regorafenib against colorectal and gastric cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5510.

Our Laboratory was established in 1994 at Univ. of Pennsylvania. Lab members demonstrated initial competencies by performing cell culture, western blots, immunofluorescence, and flow cytometry showing induction of p53/p21(WAF1) in cells treated with chemotherapy. Years later, our Laboratory of Translational Oncology & Experimental Cancer Therapeutics moved to Penn State Univ., Fox Chase Cancer Center/Temple Univ. and then Brown Univ. By 2020, with desire for inclusiveness (everyone succeeds), scientific rigor/reproducibility mandated by NIH, and as a training and mentoring activity (lab scientists/trainees/students mentoring others at High School level and beyond), we established a process for onboarding and training new cancer researchers. By Fall of 2022, there were 17 current Brown University undergraduate students (10 receiving research credit and 7 not receiving credit), HS students, 7 graduate students (PhD, masters, MD/PhD), and 6 medical students working with collaborating faculty at our laboratory at Brown’s Legorreta Cancer Center. After completion of biosafety training, and required trainings such as by IACUC, new lab members complete basic competencies in cell culture, cell viability, and western blot analysis that include technical, presentation quality output, and quantitative/statistical rigor to satisfy current standards for journal publication. For cell culture this includes pathogen free conditions, authentication, attention to details of routine procedures, documentation of morphology, freezing, thawing, passaging, seeding density, and managing cell populations to not run out of cells. Cell viability assessment includes attention to culture conditions, synergy analysis, data robustness, and presentation, and for western blots attention to quality of blots, protein quantification, loading, labeling, antibody specificity and sensitivity controls, presentation at 2022 standards, conventions for splicing, and issues with reproducibility including biological replicates, and generalizability. Additional and advanced competencies include RT-PCR, long-term colony assays, 3-D cultures (spheroids, organoids), transfection (overexpression, knockdown, CRISPR), co-culture and triculture with immune cells and fibroblasts, cytokine profiling, in vivo studies, in vivo imaging, immunohistochemistry, flow cytometric analysis, single cell techniques, viral infection, circulating tumor cell isolation, blood immune and cytokine analysis, and work with transgenic organoids and inducible cancer predisposing alleles. Modeling the tumor microenvironment, relevance to human cancer and translational directions are emphasized. Shared online lab resources, protocols, practices, videos, and manuscripts are available for lab members. The framework herein may be of interest to others involved in similar training programs. Citation Format: Wafik S. El-Deiry, Andrew George, Francesca Di Cristofano, Praveen Srinivasan, Lindsey Carlsen, Kelsey E. Huntington, Arielle De La Cruz, Leiqing Zhang, Marina Hahn, Shuai Zhao, Attila Seyhan, Bradley D. DeNardo, Aaron W. Maxwell, Dae Hee Kim, Alex Raufi, Hina Khan, Stephanie L. Graff, Don S. Dizon, Christopher Azzoli, Abbas E. Abbas, Roxanne Wood, Rishi R. Lulla, Howard P. Safran, Benedito A. Carneiro, Arunasalam Navaraj, Xiaobing Tian, Shengliang Zhang, Lanlan Zhou. Inclusive basic and advanced translational laboratory research competencies for research in cancer biology and therapeutics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4185.

Hypoxia is a common characteristic in solid cancers. Hypoxia-inducible factors (HIFs) are involved in various aspects of cancer, such as angiogenesis, metastasis and therapy resistance. Targeting the HIF pathway has been regarded as a challenging but promising strategy in cancer treatment with recent FDA approval of a HIF2α-inhibitor. During the past several decades, numerous efforts have been made to understand how HIFs participate in cancer development and progression along with how HIF signaling can be modulated to achieve anti-cancer effect. In this chapter, we will provide an overview of the role of hypoxia and HIFs in cancer, summarize the oxygen-dependent and independent mechanisms of HIF-1α regulation, and discuss emerging approaches targeting hypoxia and HIF signaling which possess therapeutic potential in cancer. We will emphasize on two signaling pathways, involving cyclin-dependent kinases (CDKs) and heat shock protein 90 (HSP90), which contribute to HIF-1α (and HIF-2α) stabilization in an oxygen-independent manner. Through reviewing their participation in malignant progression and the potential targeting strategies, we discuss the non-canonical approaches to target HIF signaling in cancer therapy.

HIF-1α is the main mediator of hypoxic response in cells and contributes to angiogenesis, metastasis and therapy resistance in cancer. The canonical pathway of HIF-1α degradation in normoxia is through the Von Hippel-Lindau (VHL) E3 ubiquitin ligase. We previously found that knockdown of CDK4 decreased HIF-1α expression in a VHL-independent manner. To explore the mechanism of CDK4-induced HIF-1α stabilization, we performed a proteomic analysis on HIF-1α-interacting proteins with palbociclib treatment, a bioinformatic prediction of HIF-1α-targeting E3 ubiquitin ligases and identified SMAD specific E3 ubiquitin protein ligase 2 (Smurf2) to be involved in a non-canonical HIF-1α regulation mechanism. Overexpression of Smurf2 enhanced the ubiquitination of HIF-1α and reduced expression of HIF-1α in HCT116 colorectal cancer cells. Smurf2 overexpression downregulated HIF-1α target gene SLC2A1 but not HIF1A mRNA level in hypoxia. Knockdown of SMURF2 increased HIF-1α expression in normoxia even when CDK4 was knocked-down. TCGA analysis showed correlation between high SMURF2 mRNA and better prognosis in kidney renal clear cell carcinoma (KIRC). In KIRC, VHL loss is a frequent mutational event that leads to high constitutive expression of HIF-1α and HIF-2α. Thus, we investigated a role for Smurf2 in HIF regulation in this cancer type. Overexpression of Smurf2 suppressed HIF-1α expression in RCC4 VHL-deficient clear cell renal cancer line in normoxia. Knockdown of SMURF2 increased HIF-1α expression in RCC4 cells. Treatment of RCC4 cells with Smurf2 inhibitor Heclin also increased HIF-1α expression in normoxia. HIF-2α expression was reduced upon Smurf2 overexpression in RCC4 cells under normoxia and in SW480 colorectal cancer cells under hypoxia. In summary, Smurf2 mediates VHL-independent HIF-1α degradation, which provides new insights for HIF-1α targeting in cancers especially with VHL deficiency, and targeting of Smurf2 to increase HIF activity may have functional consequences in non-neoplastic disorders. Citation Format: Shuai Zhao, Wafik S. El-Deiry. HIF-1α regulation by Smurf2-mediated protein degradation in a VHL-independent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 120.

The major adaptive response to hypoxia involves hypoxia-inducible factor HIF-1α which is regulated by von Hippel Lindau (VHL) E3 ligase. We previously observed a stabilization of HIF-1α by cyclin-dependent kinases CDK1 and CDK4/6 that is independent of VHL, hypoxia or p53, and found that CDK4/6 inhibitors destabilize HIF-1α under normoxia and hypoxia. To further investigate the molecular mechanism of HIF-1α destabilization by CDK1 or CDK4/6 inhibitors, we performed a proteomic screen on immunoprecipitated HIF-1α from hypoxic colorectal cancer cells that were either untreated or treated with CDK1 inhibitor Ro3306 and CDK4/6 inhibitor palbociclib. Our proteomics screen identified a number of candidates that were enriched in palbociclib-treated hypoxic cells including SMAD specific E3 ubiquitin protein ligase 2 (Smurf2). We also identified a HIF-1α peptide that appeared to be differentially phosphorylated after palbociclib treatment. Gene knockdown of SMURF2 increased basal expression of HIF-1α even in the presence of Ro3306 or two different CDK4/6 inhibitors, palbociclib and abemaciclib. Overexpression of Smurf2 inhibited expression of HIF-1α and enhanced HIF-1α ubiquitination in normoxia. Proteasome inhibitor MG-132 partially rescued HIF-1α expression when Smurf2 was overexpressed. Smurf2 overexpression also inhibited HIF-1α expression level in two other cell lines, SW480 and VHL-deficient RCC4. Overexpression of SMURF2 mRNA is correlated with improved disease-free survival and overall survival in clear cell renal cell cancer. Our results unravel a previously unknown mechanism involving Smurf2 for HIF-1α destabilization in CDK4/6 inhibitor-treated cells, thereby shedding light on VHL-independent HIF-1α regulation.

A prevalent characteristic of solid tumors is intra-tumoral hypoxia. Hypoxia-inducible factor 1α (HIF1α) predominantly mediates the adaptive response to O 2 oscillation and is linked to multiple malignant hallmarks. Here we describe a strategy to robustly target HIF1α by dual inhibition of CDK(s) and heat shock protein 90 (HSP90). We show that CDK1 may contribute to HSP90-mediated HIF1α stabilization. CDK1 knockdown enhances the decrease of HIF1α by HSP90 inhibition. Dual inhibition of CDK1 and HSP90 significantly increases apoptosis and synergistically inhibits cancer cell viability. Similarly, targeting CDK4/6 using FDA-approved inhibitors in combination with HSP90 inhibition shows a class effect on HIF1α inhibition and cancer cell viability suppression not only in colorectal but also in various other cancer types, including Rb -deficient cancer cells. Dual inhibition of CDK4/6 and HSP90 suppresses tumor growth in vivo. In summary, combined targeting of CDK(s) (CDK1 or CDK4/6) and HSP90 remarkably inhibits the expression level of HIF1α and shows promising anti-cancer efficacy with therapeutic potential.

Hypoxia is an important phenomenon in solid tumors that contributes to metastasis, tumor microenvironment (TME) deregulation, and resistance to therapies. The receptor tyrosine kinase AXL is an HIF target, but its roles during hypoxic stress leading to the TME deregulation are not well defined. We report here that the mammary gland-specific deletion of Axl in a HER2+ mouse model of breast cancer leads to a normalization of the blood vessels, a proinflammatory TME, and a reduction of lung metastases by dampening the hypoxic response in tumor cells. During hypoxia, interfering with AXL reduces HIF-1α levels altering the hypoxic response leading to a reduction of hypoxia-induced epithelial-to-mesenchymal transition (EMT), invasion, and production of key cytokines for macrophages behaviors. These observations suggest that inhibition of Axl generates a suitable setting to increase immunotherapy. Accordingly, combining pharmacological inhibition of Axl with anti-PD-1 in a preclinical model of HER2+ breast cancer reduces the primary tumor and metastatic burdens, suggesting a potential therapeutic approach to manage HER2+ patients whose tumors present high hypoxic features.Copyright © 2021 the Author(s). Published by PNAS. PMID: 34266948

Many primary tumours have low levels of molecular oxygen (hypoxia), and hypoxic tumours respond poorly to therapy.Pan-cancer molecular hallmarks of tumour hypoxia remain poorly understood, with limited comprehension of its associations with specific mutational processes, non-coding driver genes and evolutionary features.Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2658 cancers across 38 tumour types, we quantify hypoxia in 1188 tumours spanning 27 cancer types.Elevated hypoxia associates with increased mutational load across cancer types, irrespective of underlying mutational class.The proportion of mutations attributed to several mutational signatures of unknown aetiology directly associates with the level of hypoxia, suggesting underlying mutational processes for these signatures.At the gene level, driver mutations in TP53, MYC and PTEN are enriched in hypoxic tumours, and mutations in PTEN interact with hypoxia to direct tumour evolutionary trajectories.Overall, hypoxia plays a critical role in shaping the genomic and evolutionary landscapes of cancer.

As solid tumors outgrow their oxygen supply, regions with abnormal vasculature become poorly oxygenated, known as intratumoral hypoxia. Hypoxia-inducible factor 1α (HIF1α) is the main mediator to hypoxia and promotes angiogenesis, cell survival as well as cancer metastases. Overexpression of HIF1α is correlated with poor prognosis in many malignancies such as colorectal cancers. Targeting HIF1α is a potential strategy for cancer therapy. We have previously shown that cyclin-dependent kinase 1 (CDK1) stabilizes HIF1α through direct phosphorylation of its Ser668 residue in a Von Hippel-Lindau (VHL)-independent manner both under hypoxia and at G2/M under normoxia (Warfel et al., 2013). It has been acknowledged that the heat shock protein 90 (HSP90) is also a VHL-independent HIF1α stabilizer (Isaacs JS et al., 2002). We found that CDK1 may contribute to HSP90-mediated HIF1α stabilization. Combination treatment of HSP90 inhibition and CDK1 knockdown reduces HIF1α expression in hypoxia more robustly than either single treatment. Dual inhibition of CDK1 and HSP90 synergistically decreases cell viability in colorectal cancer cells and suppresses colony formation. We have observed that CDK4 plays a role in HIF1α stabilization. Using the FDA-approved CDK4/6 inhibitors (palbociclib & abemaciclib), we observed that dual inhibition of CDK4 and HSP90 synergistically inhibits cancer viability in colorectal cancer cell lines (eg. HCT116, SW480, DLD1) under both normoxia and hypoxia. Multiple HSP90 inhibitors have been tested, including ganetespib, onalespib, XL888 and TAS116, which indicates such combinational inhibition as a class effect. As expected, the combination treatment of CDK4 knockdown/inhibition and HSP90 inhibition is able to reduce the level of HIF1α and suppress cell viability in various cancer cell lines (eg. colorectal, glioblastoma, breast, and prostate cancers). The combination treatment induces PARP cleavage, indicating activated apoptosis. Interestingly, we found that knockdown of RB does not affect the response of HCT116 or SW480 cells to the combination treatment with abemaciclib and TAS116, although RB deficiency contributes to a relative resistance to CDK4/6 inhibitor monotherapy. Combination of abemaciclib plus TAS116 inhibits cell viability in Rb-deficient cells (eg. H1048 and Saos2). Hypoxia sensitizes the Rb-deficient MDA-MB-468 breast cancer cell line to abemaciclib treatment. Our findings suggest a therapeutic potential for utilizing the combination of CDK4 and HSP90 inhibitors in cancer treatment. Citation Format: Shuai Zhao, Lanlan Zhou, David T. Dicker, Wafik S. El-Deiry. Anti-tumor effect and HIF1α inhibition by combining CDK4 inhibitor with HSP90 inhibitor in various cancer types including Rb-deficient tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3564.

Most cancers harbor intra-tumoral hypoxia which promotes tumor progression and therapy resistance. Hypoxia-inducible factor 1α (HIF1α) mediates an adaptive response to hypoxia and contributes to multiple cancer hallmarks. We describe cancer therapeutic targeting of HIF1α by combination of CDK4/6 inhibitors (CDK4/6i) and heat-shock protein 90 inhibitors (HSP90i). CDK1 contributes to HSP90-mediated HIF1α stabilization whereas CDK1-knockdown enhances HIF1α reduction by HSP90i. Dual CDK1- and HSP90-inhibition increases apoptosis and synergistically inhibits cancer cell viability. To translate our findings, we use FDA-approved CDK4/6i in combination with HSP90i to reduce HIF1α expression and suppress viability of multiple cancer cell types, including Rb-deficient cancer cells. Overexpression of HIF1α 668E partially rescues the cell viability inhibition by combination CDK4/6i and HSP90i treatment under hypoxia. CDK4/6i and HSP90i suppresses tumor growth in vivo . Thus, combined targeting of CDK4/6 and HSP90, through a drug class effect, inhibits HIF1α and shows preclinical anti-cancer therapeutic efficacy, including with Rb-deficiency.

ONC201/TIC10 is a promising anticancer agent that upregulates cytotoxic TRAIL pathway signaling in cancer cells. Preliminary clinical data indicates ONC201 induces clinical benefit in a subset of patients with histone H3 K27M glioma, among other tumors. Since H3 K27M mutation reduces levels of H3K27 di- and tri-methylation and in turn alters the expression of genes by epigenetic modulation, we investigated ONC201 effects on epigenetic regulation in cancer. We treated the colorectal cancer cell line HCT116 with the histone deacetylase inhibitor entinostat or ONC201. Chromatin immunoprecipitation (ChIP) was performed with anti-histone H3 (acetyl K9) antibody using lysates from drug-treated tumor cells. Immunoprecipitated DNA was probed by PCR using primers across the promoter of p21 (WAF1; CDKN1A). The results indicate that both ONC201 and entinostat induce the acetylation of histone H3 binding within the p21 promoter. We further observed that ONC201 plus entinostat have synergistic effects in this p21 promoter acetylation activity. Activation of p21 gene expression by enhancing histone acetylation may be relevant in cancer suppression by ONC201. Citation Format: Yiqun Zhang, Lanlan Zhou, Shengliang Zhang, Marie D. Ralff, Shuai Zhao, Philip H. Abbosh, Rahmat Sidker, Wafik S. El-Deiry. ONC201 induces acetylation of histone binding within the p21 (CDKN1A) gene promoter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5183.