Retinoblastoma Tumor Suppressor Research Articles

Pathogenic Mechanisms Activated by NSD2 Translocations in Multiple Myeloma

The NSD2 gene is overexpressed in multiple cancers and is associated with cancer cell proliferation, invasion, and metastasis. NSD2 gene is universally present in multiple myeloma (MM) patients with t(4;14) (p16;q32) translocation, a high-risk subtype. NSD2 is characterized by different breakpoints. NSD2Δ3 breakpoint is associated with shorter survival than other breakpoints. NSD2Δ3 contains non-coding RNA ACA11 within intron 18. Additionally, 80% of t(4;14) MM patients lack the retinoblastoma tumor suppressor gene (RB1). We hypothesize that ACA11 cooperates with RB1 loss in MM pathogenesis. Also, we hypothesize that NSD2Δ3 increases protein expression driving myeloma growth. To overexpress NSD2WT, snoACA11, and NSD2Δ3 in cells, we cloned NSD2WT, and NSD2Δ3 plasmid constructs in a murine stem cell virus (MSCV)-IRES- GFP vector. We isolated primary B cells from RB1 wild-type and knock-out mice by CD43 microbeads and transduced those cells using a virus containing ACA11 and NSD2WT. ACA11 overexpression significantly increased B cell proliferation in Rb1dKO mice. This hyperproliferative phenotype is induced by reactive oxygen species (ROS) levels. However, we did not find a further increase in B cell proliferation with co-overexpression of NSD2WT and ACA11 in Rb1dKO mice. We demonstrated increased protein stability by finding higher NSD2Δ3 protein expression compared to NSD2WT protein expression in HEK293T cells. These results support our hypothesis and demonstrate oncogenic role of ACA11 in t(4;14) MM. Future work will be done to determine the cell biological effects of NSD2Δ3 and establish its role in myeloma pathogenesis. Completion of this study will advance understanding of this epigenetic NSD2 gene and potentially develop targeted drugs for NSD2, providing more individualized treatment options for these patients. University of Iowa Department of Internal Medicine. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Regulation of Molecular Biomarkers Associated with the Progression of Prostate Cancer.

Androgen receptor signaling regulates the normal and pathological growth of the prostate. In particular, the growth and survival of prostate cancer cells is initially dependent on androgen receptor signaling. Exposure to androgen deprivation therapy leads to the development of castration-resistant prostate cancer. There is a multitude of molecular and cellular changes that occur in prostate tumor cells, including the expression of neuroendocrine features and various biomarkers, which promotes the switch of cancer cells to androgen-independent growth. These biomarkers include transcription factors (TP53, REST, BRN2, INSM1, c-Myc), signaling molecules (PTEN, Aurora kinases, retinoblastoma tumor suppressor, calcium-binding proteins), and receptors (glucocorticoid, androgen receptor-variant 7), among others. It is believed that genetic modifications, therapeutic treatments, and changes in the tumor microenvironment are contributing factors to the progression of prostate cancers with significant heterogeneity in their phenotypic characteristics. However, it is not well understood how these phenotypic characteristics and molecular modifications arise under specific treatment conditions. In this work, we summarize some of the most important molecular changes associated with the progression of prostate cancers and we describe some of the factors involved in these cellular processes.

Uncovering Novel Protein Partners of Inducible Nitric Oxide Synthase in Human Testis.

Peroxidative damage to human spermatozoa has been shown to be the primary cause of male infertility. The possible role of nitric oxide (NO) in affecting sperm motility, capacitation, and acrosome reaction has been reported, too. The overproduction of NO by the enzyme inducible nitric oxide synthase (iNOS) could be responsible as it has been implicated in the pathogenesis of many diseases. There have been many studies on regulating iNOS function in various tissues, especially by protein-protein interaction; however, no study has looked for iNOS-interacting proteins in the human testis. Here, we have reported the identification of two proteins that interact with iNOS. We initially undertook a popular yeast two-hybrid assay to screen a human testis cDNA library in yeast using an iNOS-peptide fragment (amino acids 181-335) as bait. We verified our data using the mammalian chemiluminescent co-IP method; first, employing the same peptide and, then, a full-length protein co-expressed in HEK293 cells in addition to the candidate protein. In both cases, these two protein partners of iNOS were revealed: (a) sperm acrosome-associated 7 protein and (b) retinoblastoma tumor-suppressor binding protein.

Subscaling of a cytosolic RNA binding protein governs cell size homeostasis in the multiple fission alga Chlamydomonas.

Coordination of growth and division in eukaryotic cells is essential for populations of proliferating cells to maintain size homeostasis, but the underlying mechanisms that govern cell size have only been investigated in a few taxa. The green alga Chlamydomonas reinhardtii (Chlamydomonas) proliferates using a multiple fission cell cycle that involves a long G1 phase followed by a rapid series of successive S and M phases (S/M) that produces 2n daughter cells. Two control points show cell-size dependence: the Commitment control point in mid-G1 phase requires the attainment of a minimum size to enable at least one mitotic division during S/M, and the S/M control point where mother cell size governs cell division number (n), ensuring that daughter distributions are uniform. tny1 mutants pass Commitment at a smaller size than wild type and undergo extra divisions during S/M phase to produce small daughters, indicating that TNY1 functions to inhibit size-dependent cell cycle progression. TNY1 encodes a cytosolic hnRNP A-related RNA binding protein and is produced once per cell cycle during S/M phase where it is apportioned to daughter cells, and then remains at constant absolute abundance as cells grow, a property known as subscaling. Altering the dosage of TNY1 in heterozygous diploids or through mis-expression increased Commitment cell size and daughter cell size, indicating that TNY1 is a limiting factor for both size control points. Epistasis placed TNY1 function upstream of the retinoblastoma tumor suppressor complex (RBC) and one of its regulators, Cyclin-Dependent Kinase G1 (CDKG1). Moreover, CDKG1 protein and mRNA were found to over-accumulate in tny1 cells suggesting that CDKG1 may be a direct target of repression by TNY1. Our data expand the potential roles of subscaling proteins outside the nucleus and imply a control mechanism that ties TNY1 accumulation to pre-division mother cell size.

P53 and Rb Aberrations in Small Cell Lung Cancer (SCLC): From Molecular Mechanisms to Therapeutic Modulation.

The genes coding for the tumor suppressors p53 and retinoblastoma (Rb) are inactivated in the vast majority of small cell lung cancer (SCLC) tumors. Data support the notion that these two deleterious genetic events represent the initial steps in the development of SCLC, making them essential for a lung epithelial cell to progress toward the acquisition of a malignant phenotype. With the loss of TP53 and RB1, their broad tumor suppressive functions are eliminated and a normal cell is able to proliferate indefinitely, escape entering into cellular senescence, and evade death, no matter the damage it has experienced. Within this setting, lung epithelial cells accumulate further oncogenic mutations and are well on their way to becoming SCLC cells. Understanding the molecular mechanisms of these genetic lesions and their effects within lung epithelial cells is of paramount importance, in order to tackle this aggressive and deadly lung cancer. The present review summarizes the current knowledge on p53 and Rb aberrations, their biological significance, and their prospective therapeutic potential, highlighting completed and ongoing clinical trials with agents that target downstream pathways.

Knowledge of Cervical Cancer and Attitude Toward Cervical Cancer Screening Among the First-Year School of Midwifery Students in FMC Nguru, North-Eastern Nigeria

Cervical cancer occurs due to Infection by high-risk human papillomavirus (HPV) and subsequent integration of the HPV genome into the host chromosome of cervical epithelial cells. The viral proto-oncogenes, mainly E6 and E7, are responsible for the initial changes in the epithelial cells of the transformation zone of the cervix. The viral proteins inactivate two main tumor suppressor proteins, p53, and retinoblastoma (pRb). Inactivation of these host proteins disrupts both the DNA repair mechanisms and apoptosis, leading to rapid cell proliferation. Cervical cancer is the 4th most common cancer among women worldwide, it ranked 14th among all cancers worldwide. There are 500,000 new cases of cancer of the cervix annually, and about 250,000 mortalities all over the world from cervical cancer. About 80% of all these cases occur in low-income countries. In Nigeria, there are 12,075 new cases of cervical cancer and 7,968 deaths due to cervical cancer per year. The study was a cross-sectional type aimed at exploring the knowledge of the participants on cancer of the cervix and their attitude toward cervical cancer screening using a semi-structured questionnaire in FMC Nguru. Results: The total number of participants was 29(n=29). The mean age of the respondents was 20.9 +/-2. 8SD. Concerning the knowledge of cervical cancer among the participants, 27(93.1%) heard of cervical cancer. Most of the respondents do not know the cause of cervical cancer, as only 10(34.5%) know that cervical cancer is associated with HPV. The risk factors for cervical Cancer are not known by most of the respondents as 14(48.3%) agreed that having a relative who had the disease is a risk factor, while only 10(34.5%) agreed that HPV is a cause of the pathology. There was poor knowledge regarding the presentation of cancer of the cervix, abnormal vaginal bleeding is the most popular way of presentation known by 13(44.8%) of the respondents while only 8(27.6%) know that cervical cancer can present with bleeding after sexual intercourse, five of the respondents (17.2%) showed smelling vaginal discharge as the mode of presentation, dysuria by only 2(6.9%). A good number of the participants know about cervical screening 26(89.7%). Twenty-eight respondents (96.6%) are willing to go and have themselves screened for cervical cancer if they are asked to do so, and 27(93.1%) will advise some other people to go for the screening. A good number of the respondents, 27(93.1%) are aware that cancer of the cervix can be prevented. Conclusion: The students had a good awareness of cervical cancer and cancer of the cervix screening and had a positive attitude toward having themselves screened whenever the chance was available, and also had a positive attitude toward being willing to convince other people to go for the screening, but demonstrated less knowledge on cervical cancer causes, risk factors and mode of presentation. Hence there is a window of opportunity to deliver more health education to the students and by extension to the larger population.

Use of Pharmacokinetic and Pharmacodynamic Data to Develop the CDK4/6 Inhibitor Ribociclib for Patients with Advanced Breast Cancer.

Ribociclib is an orally bioavailable, selective cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor. CDK4/6inhibition by ribociclib leads to retinoblastoma tumor suppressor protein (Rb) reactivation, thereby restoring Rb-mediated cell cycle arrest. Ribociclib is approved for the treatment of patients with hormone receptor-positive/human epidermal growth factor receptor-2-negative (HR+/HER2-) advanced breast cancer (ABC), at the dose of 600mg once daily (QD) during cycles of 21days on/7days off, with optional dose reduction to 400 mgand 200mg. Ribociclib is rapidly absorbed with a median time to reach maximum plasma concentration of 2.4h, mean half-life of 32.0h and oral bioavailability of 65.8% at 600mg. It is eliminated mainly by hepatic metabolism (~84% of total elimination), mostly by cytochrome P450 (CYP) 3A4. Age, body weight, race, baseline Eastern Cooperative Oncology Group status, food, mild hepatic impairment, mild-to-moderate renal impairment, proton pump inhibitors, and combination partners (non-steroidal aromatase inhibitors or fulvestrant) have no clinically relevant impact on ribociclib exposure. Ribociclib inhibits CYP3A at 600mg leading to increased exposure of CYP3A substrates. Strong CYP3A inhibitors or inducers increase or decrease, respectively, ribociclib exposure. Exposure-safety and exposure-efficacy analyses support the clinical benefit of the 600mg QD starting dose, with potential individualized dose reductions to 400 mgand 200mg for effective management of the adverse events neutropenia and QTcF interval prolongation, while maintaining efficacy, in patients with HR+/HER2- ABC. Overall, these clinical pharmacology data informed ribociclib dose justification and clinical development, as well as its prescribing information for clinical use in advanced breast cancer patients.

Abstract PR007: The combination of TRIP13 and Aurora kinase A inhibition caused cell cycle specific DNA damage and death in Rb-deficient cancers

Abstract Many diverse, common, and lethal cancer types have inactivation of the retinoblastoma (RB1, Rb) tumor suppressor pathway including cancers caused by the human papillomavirus (HPV), which causes >5% of all cancers worldwide. Despite therapeutic advances, advanced Rb-deficient cancers are still lethal with therapy-related adverse effects. Moreover, there are no therapies that uniquely target HPV-driven or Rb-deficient cancers. Therefore, biomarker-selected, targeted therapy for Rb-deficient cancers represents both a substantial unmet need and a significant translational knowledge gap. Rb-deficient cancer cells depend on Aurora A kinases to bypass the spindle assembly checkpoint (SAC) and survive due to overexpression of the mitotic checkpoint protein Mad2. Thyroid hormone receptor interactor 13 (TRIP13) catalyzes the ATP-dependent conversion of Mad2 from its active to inactive form, which turns off the SAC. Our lab discovered that the combination of Aurora A inhibition with alisertib plus TRIP13 depletion induced extensive apoptosis selectively in Rb-deficient cancer cells. Moreover, our results demonstrated that the synthetic lethality between Aurora A and TRIP13 reflects mitotic catastrophe. The combination led to highly abnormal mitoses in Rb-deficient cancer cells, evidenced by unaligned, dispersed chromosomes, mitotic spindle defects, and G2/M arrest. Moreover, live cell imaging showed that alisertib treatment increased the duration of first mitoses and induced mitotic cell death, which was even more pronounced in second mitotic events. The combination resulted in both prolonged mitotic arrest and enhanced lethality, indicated by increased mitotic cell death after the first mitoses and interphase cell death following the first mitotic arrest. Furthermore, using degron tagging to rapidly degrade TRIP13, we showed that TRIP13 depletion led to transient mitotic arrest in cells that were in late G2, supporting the role of TRIP13 in regulating the SAC and mitotic progression. Since prolonged mitotic arrest is known to cause DNA damage and both Aurora A and TRIP13 are known to be involved in the DNA damage response, we also tested the effect of the combination treatment on DNA damage. Treating Rb-deficient cancer cells with the combination resulted in robust increases in gamma-H2AX, pTIF1 beta (KAP1, TRIM28), and DNA PKcs. The lethal effects of the combination required mitotic entry as concurrent CDK1 inhibition with RO-3306 prevented apoptosis and DNA damage induced by the combination. The selective effect of the combination in Rb-deficient cancer cells may have a wide therapeutic index resulting in less toxic and more effective therapy. Additionally, targeting mitosis may contribute to a cancer-specific effect due to cancer cells’ high proliferation rate. Our study suggests that targeting TRIP13 and Aurora A led to DNA damage and death specifically during mitosis in Rb-deficient cells. This work has potential to establish a foundation for future clinical trials and may change current clinical practice paradigms. Citation Format: Lacin Yapindi, Soma Ghosh, Tuhina Mazumdar, Faye M. Johnson. The combination of TRIP13 and Aurora kinase A inhibition caused cell cycle specific DNA damage and death in Rb-deficient cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr PR007.

Abstract C062: Defining the interplay between PARP and the RB/E2F axis in prostate cancer

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related death in American men. Non-Hispanic Black men are 1.8 times more likely to develop PCa and 2.3 times more likely to die from PCa. Tumor suppressor retinoblastoma (RB1) is frequently deleted in PCa. RB represses the transcriptional activity of E2Fs and the phosphorylation of RB by CDK4/6 results in dissociation of the complex and a subsequent increase in E2F transcriptional activity, leading to cell cycle progression. RB loss is associated with tumor aggressiveness occurring in 10-15% of castration-resistant prostate cancer (CRPC) tumors and corresponds to a decrease in overall survival. RB depletion leads to increased expression of DNA repair proteins due to elevated transcriptional activity of E2F1. PARP-1 is an enzyme that plays a role in DNA repair. Elevated PARP-1 enzymatic activity is associated with PCa progression and correlates with poor clinical outcomes in CRPC. E2F1 transcriptional activity is supported by PARP enzymatic activity, however the consequences of the functional interaction between the RB/E2F axis and PARP remains an open line of inquiry. Defining the interplay between PARP and RB/E2F is likely to contribute to the identification of novel treatment strategies and/or biomarker response. Canonically, E2F1-3 are transcription factors that positively regulate gene expression programs associated with pro-proliferative signals. Prior research by other groups has shown that E2F target genes expression is elevated in tumors excised from Black men compared to Asian and White men. There was an increase in germline RAD51 mutations observed. RAD51 is an E2F target gene involved in DDR, therefore attenuating PARP enzymatic activity may be a potential mode of targeting this axis. These data suggest that there may be differential DNA damage response (DDR) in African American men driven by aberrations in the RB/E2F signaling axis, however the mechanism is yet to be fully explored. Analysis of publicly available patient datasets were performed and demonstrate that RB1 deletions is more frequent than RB1/BRCA2 co-deletion in metastatic PCa. Isogenic models of RB1 knockdown were used to evaluate the impact of RB loss on PARP enzymatic activity. PARP-1 mRNA and PARP-1 protein are elevated upon RB1 depletion with a concomitant increase in PARP activity. Growth curve assays were conducted to examine the biological response to PARP inhibition in the isogenic models. In hormone-depleted conditions, results suggest a differential response between RB depleted models compared to the control when challenged with PARP inhibition. Furthermore, an IC50 dose of Olaparib elicits differential PARP enzymatic response in the RB1 depleted model. RB1 loss is associated with an enrichment in E2F1 binding at the PARP-1 locus. Future studies have been designed to determine the impact of the RB/E2F pathway alterations on downstream consequences PARP enzymatic function and establish the contribution of PARP and the RB/E2F axis on racial disparity in PCa. Citation Format: Latese Evans. Defining the interplay between PARP and the RB/E2F axis in prostate cancer [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C062.

Abstract A022: CDK2 inhibition demonstrates synthetic lethality in SCLC through apoptotic induction

Abstract Small cell lung cancer (SCLC), a malignant thoracic neoplasm genetically defined by dysregulation of the Retinoblastoma (RB1) gene and tumor suppressor protein 53 (TP53), remains one of the world’s deadliest tumors with a 5-year overall survival rate ranging from 5% to 10%. Despite numerous clinical trials and recent FDA approval of immune checkpoint inhibitors, the prognosis for SCLC has not significantly improved in decades. There remains a strong need for new therapeutic approaches. In this study, we found that the combined effect of dysregulation or deletion of RB1 and TP53, as is found in the majority of SCLC, results in sensitivity to CDK2 inhibition or ablation. This relationship is reflected in the DepMap database, with cell lines harboring deleterious mutations in RB1 demonstrating markedly higher susceptibility to CDK2 inhibition. This relationship is surprising given CDK2’s established role in suppressing the growth inhibition activity of RB and CDK2’s ability to thus facilitate G1/S transition. To validate this discovery, we evaluated the effects of CDK2 inhibition on a panel of SCLC cell lines. ARTS-021 is a potent and highly selective orally administered inhibitor of CDK2 currently in clinical development. In contrast to CCNE1 amplified cells, which undergo G1 arrest upon ARTS-021 treatment, SCLC cells treated with ARTS-021 exhibited minimal G1 accumulation. Instead, ARTS-021 treatment induced apoptosis in SCLC cell lines with loss of functional RB1 and TP53, as demonstrated by a dose-dependent increase in Caspase 3/7 activity and the accumulation of cleaved PARP. This phenotype was further demonstrated to be CDK2 dependent though generation of stable cell lines expressing a dox-inducible shRNA targeting CDK2. Combination studies further demonstrated that this apoptotic phenotype can be enhanced synergistically through inhibition of the anti-apoptotic proteins BCL-2 and BCL-xL with clinical stage inhibitor Navitoclax. In addition, combinations with currently approved standard of care chemotherapies were tested both in vitro and in vivo, and demonstrated potent combinatorial activity. These findings suggest that the clinical development of CDK2 inhibitors for SCLC may represent a novel therapeutic option for this difficult to treat malignancy. Citation Format: Nathan Schomer, Jinhong Chen, Wei Pang, Tingru Zhou, Xipan Liu, Wenhen Liang, Yali Guo, Dai Cheng, Fang Li, Jiaqi Liang, Yaoyu Chen, Qing Sheng. CDK2 inhibition demonstrates synthetic lethality in SCLC through apoptotic induction [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A022.

Cyclin-dependent kinase 4/6 inhibitors - Ribociclib - Induced ILD/Pneumonitis - The importance of early diagnosis: A case report

CDK4/6 inhibitors have demonstrated significant clinical activity in cancer patients. CDK4/6 inhibitors (palbociclib, ribociclib, and abemaciclib) and endocrine therapy have been approved for the treatment of advanced breast cancer in patients with HR+ and HER2-negative (HER2-) subtypes. The canonical mechanism underlying the activity of CDK4/6 inhibitors is the inhibition of retinoblastoma tumor suppressor protein phosphorylation, which prevents cancer cell proliferation. Common CDK4/6 inhibitors used to treat breast cancer include palbociclib, ribociclib, and abemaciclib, and have been utilized with great success in the treatment of hormone receptor-positive breast cancers and are undertaking testing for numerous other types of tumors. CDK4/6 inhibitors may induce nausea, vomiting, diarrhea, fatigue, alopecia, and myelosuppression symptoms such as leukopenia, neutropenia, and thrombocytopenia mucositis, hepatic toxicities (transaminitis), and cardiotoxicity (QTc prolongation, arrhythmias, novel hypertension, atrial fibrillation, myocardial infarction, pericardial effusion, cardiac failure). Despite a favorable toxicity profile in pivotal studies, increased rates of use in clinical practice may be associated with rare but fatal toxicity, such as pulmonary injury. The FDA has issued a warning regarding a rare but severe pulmonary inflammation that may be caused by the use of CDK4/6 inhibition in advanced breast cancer patients. This article displays a rare case of ribociclib-induced ILD/pneumonitis in a 53-year-old female with metastatic breast cancer

Unveiling the molecular structure and role of RBBP4/7: implications for epigenetic regulation and cancer research.

Retinoblastoma-binding protein (RBBP) family is a class of proteins that can interact with tumor suppressor retinoblastoma protein (pRb). RBBP4 and RBBP7 are the only pair of homologous proteins in this family, serving as scaffold proteins whose main function is to offer a platform to indirectly connect two proteins. This characteristic allows them to extensively participate in the binding of various proteins and epigenetic complexes, indirectly influencing the function of effector proteins. As a result, they are often highlighted in organism activities involving active epigenetic modifications, such as embryonic development and cancer activation. In this review, we summarize the structural characteristics of RBBP4/7, the complexes they are involved in, their roles in embryonic development and cancer, as well as potential future research directions, which we hope to inspire the field of epigenetic research in the future.

A cell-based model for size control in the multiple fission alga Chlamydomonas reinhardtii

Understanding how population-size homeostasis emerges from stochastic individual cell behaviors remains a challenge in biology.1,2,3,4,5,6,7 The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) proliferates using a multiple fission cell cycle, where a prolonged G1 phase is followed by n rounds of alternating division cycles (S/M) to produce 2n daughters. A "Commitment" sizer in mid-G1 phase ensures sufficient cell growth before completing the cell cycle. A mitotic sizer couples mother-cell size to division number (n) such that daughter size distributions are uniform regardless of mother size distributions. Although daughter size distributions were highly robust to altered growth conditions, ∼40% of daughter cells fell outside of the 2-fold range expected from a "perfect" multiple fission sizer.7,8 A simple intuitive power law model with stochastic noise failed to reproduce individual division behaviors of tracked single cells. Through additional iterative modeling, we identified an alternative modified threshold (MT) model, where cells need to cross a threshold greater than 2-fold their median starting size to become division-competent (i.e., Committed), after which their behaviors followed a power law model. The Commitment versus mitotic size threshold uncoupling in the MT model was likely a key pre-adaptation in the evolution of volvocine algal multicellularity. A similar experimental approach was used in size mutants mat3/rbr and dp1 that are, respectively, missing repressor or activator subunits of the retinoblastoma tumor suppressor complex (RBC). Both mutants showed altered relationships between Commitment and mitotic sizer, suggesting that RBC functions to decouple the two sizers.

Histone methyltransferase activity affects metabolism in human cells independently of transcriptional regulation.

The N-terminal tails of eukaryotic histones are frequently posttranslationally modified. The role of these modifications in transcriptional regulation is well-documented. However, the extent to which the enzymatic processes of histone posttranslational modification might affect metabolic regulation is less clear. Here, we investigated how histone methylation might affect metabolism using metabolomics, proteomics, and RNA-seq data from cancer cell lines, primary tumour samples and healthy tissue samples. In cancer, the expression of histone methyltransferases (HMTs) was inversely correlated to the activity of NNMT, an enzyme previously characterised as a methyl sink that disposes of excess methyl groups carried by the universal methyl donor S-adenosyl methionine (SAM or AdoMet). In healthy tissues, histone methylation was inversely correlated to the levels of an alternative methyl sink, PEMT. These associations affected the levels of multiple histone marks on chromatin genome-wide but had no detectable impact on transcriptional regulation. We show that HMTs with a variety of different associations to transcription are co-regulated by the Retinoblastoma (Rb) tumour suppressor in human cells. Rb-mutant cancers show increased total HMT activity and down-regulation of NNMT. Together, our results suggest that the total activity of HMTs affects SAM metabolism, independent of transcriptional regulation.

Human papillomaviruses:Knowns, mysteries, and unchartered territories.

There has been an explosion in the number of papillomaviruses that have been identified and fully sequenced. Yet only a minute fraction of these has been studied in any detail. Most of our molecular research efforts have focused on the E6 and E7 proteins of "high-risk,"cancer-associated human papillomaviruses (HPVs). Interactions of the high-risk HPV E6 and E7 proteins with their respective cellular targets, the p53 and the retinoblastoma tumor suppressors, have been investigated in minute detail. Some have thus questioned if research on papillomaviruses remains an exciting and worthwhile area of investigation. However, fundamentally new insights on the biological activities and cellular targets of the high-risk HPV E6 and E7 proteins have been discovered and previously unstudied HPVs have been newly associated with human diseases. HPV infections continue to be an important cause of human morbidity and mortality and since there are no antivirals to combat HPV infections, research on HPVs should remain attractive to new investigators and biomedical funding agencies, alike.

Investigating the Potential Role of IKZF3 in HPV-Associated Carcinogenesis

HPVs cause approximately 5% of all human cancers. They encode two viral transforming proteins, E6 and E7, that are consistently expressed in these cancers. The HPV E6 and E7 proteins contribute to carcinogenesis by binding and functionally compromising multiple cellular regulatory proteins, most prominently the p53 and retinoblastoma (pRB) tumor suppressors, respectively. To discover additional cellular regulatory circuits that are dysregulated by E6 and E7 expression, we performed RNA sequencing analyses of HPV16 E6 and E7 expressing primary human genital epithelial cells. These experiments revealed that the Ikaros Zinc Finger 3 (IKZF3) transcription factor is highly upregulated in HPV16 E6/E7 expressing keratinocytes as compared to control keratinocytes. IKZF3 plays a key role in lymphocyte development, and when over-expressed or mutated, contributes to hematopoietic malignancies. IKZF3 is expressed at a very low level in normal epithelial cells, but the expression is upregulated in solid tumors, including invasive breast, cervical, and lung cancers. IKZF3 expression in these tumors causes upregulation of lymphocyte-specific genes, conferring lymphocyte-like behavior referred to as "lymphocyte mimicry," which presumably enables cancer cell survival in the bloodstream, thus potentially contributing to cancer metastasis. There is an FDA-approved drug, lenalidomide, that targets IKZF3 for degradation. Hence, we set out to determine whether IKZF3 might contribute to cervical carcinogenesis. We treated HPV16-positive SiHa and CaSki cervical cancer cells with 10 uM of lenalidomide for 12 hours. Cells were treated with equal amounts of DMSO as a negative control. Then, IKZF3 and E7 mRNA expression were determined using qRT-PCR after 12 hours of treatment. Gene expression was analyzed relative to GAPDH, and the results were analyzed using 2-way ANOVAs with Dunnett's correction for multiple comparisons. Western Blot experiments were performed to examine IKZF3, pRB, E7, and p53 (to indirectly assess E6) protein expression levels after 12 hours of lenalidomide treatment. Actin protein was used as a loading control. Each of these experiments were performed 3 times, qRT-PCR experiments were each performed in triplicate, and the results were graphed using a scientific 2-D graphing and statistics software. Although Western Blot showed that IKZF3 protein levels were significantly decreased by lenalidomide treatment, there was no change in pRB, E7, and p53 protein expression upon IKZF3 depletion. Similarly, there was no decrease in E7 mRNA expression. We showed by western blotting that targeting IKZF3 for proteasome-mediated degradation with lenalidomide effectively decreased the steady-state levels of IKZF3. However, we did not consistently observe an associated decrease in HPV gene expression. We are currently analyzing the transcriptome changes that IKZF3 depletion causes in cervical carcinoma cells to determine whether this may affect anoikis resistance.

RB loss sensitizes cells to replication-associated DNA damage after PARP inhibition by trapping.

The retinoblastoma tumor suppressor protein (RB) interacts physically and functionally with a number of epigenetic modifying enzymes to control transcriptional regulation, respond to replication stress, promote DNA damage response and repair, and regulate genome stability. To better understand how disruption of RB function impacts epigenetic regulation of genome stability and determine whether such changes represent exploitable weaknesses of RB-deficient cancer cells, we performed an imaging-based screen to identify epigenetic inhibitors that promote DNA damage and compromise the viability of RB-deficient cells. We found that loss of RB alone leads to high levels of replication-dependent poly-ADP ribosylation (PARylation) and that preventing PARylation by trapping PARP enzymes on chromatin enables RB-deficient cells to progress to mitosis with unresolved replication stress. These defects contribute to high levels of DNA damage and compromised cell viability. We demonstrate this sensitivity is conserved across a panel of drugs that target both PARP1 and PARP2 and can be suppressed by reexpression of the RB protein. Together, these data indicate that drugs that target PARP1 and PARP2 may be clinically relevant for RB-deficient cancers.

Identification of c-Met as a novel target of γ-glutamylcyclotransferase.

γ-Glutamylcyclotransferase (GGCT) is highly expressed in multiple types of cancer tissues and its knockdown suppresses the growth of cancer cells in vitro and in vivo. Although GGCT is a promising target for cancer therapy, the mechanisms underlying the antitumor effects remain unclear. The knockdown of GGCT inhibited the MEK-ERK pathway, and activated the tumor suppressor retinoblastoma gene (RB) at the protein level in cancer cell lines. c-Met was down-regulated by the knockdown of GGCT in cancer cells and its overexpression attenuated the dephosphorylation of RB and cell cycle arrest induced by the knockdown of GGCT in lung cancer A549 cells. STAT3 is a transcription factor that induces c-Met expression. STAT3 phosphorylation and its nuclear expression level were decreased in GGCT-depleted A549 and prostate cancer PC3 cells. The simultaneous knockdown of AMPK and GGCT restored the down-regulated expression of c-Met, and attenuated the dephosphorylation of STAT3 and MEK-ERK-RB induced by the knockdown of GGCT in PC3 cells. An intraperitoneal injection of a GGCT inhibitor decreased c-Met protein expression in a mouse xenograft model of PC3 cells. These results suggest that the knockdown of GGCT activates the RB protein by inhibiting the STAT3-c-Met-MEK-ERK pathway via AMPK activation.

CDK4/6-MEK Inhibition in MPNSTs Causes Plasma Cell Infiltration, Sensitization to PD-L1 Blockade, and Tumor Regression.

Malignant peripheral nerve sheath tumors (MPNST) are lethal, Ras-driven sarcomas that lack effective therapies. We investigated effects of targeting cyclin-dependent kinases 4 and 6 (CDK4/6), MEK, and/or programmed death-ligand 1 (PD-L1) in preclinical MPNST models. Patient-matched MPNSTs and precursor lesions were examined by FISH, RNA sequencing, IHC, and Connectivity-Map analyses. Antitumor activity of CDK4/6 and MEK inhibitors was measured in MPNST cell lines, patient-derived xenografts (PDX), and de novo mouse MPNSTs, with the latter used to determine anti-PD-L1 response. Patient tumor analyses identified CDK4/6 and MEK as actionable targets for MPNST therapy. Low-dose combinations of CDK4/6 and MEK inhibitors synergistically reactivated the retinoblastoma (RB1) tumor suppressor, induced cell death, and decreased clonogenic survival of MPNST cells. In immune-deficient mice, dual CDK4/6-MEK inhibition slowed tumor growth in 4 of 5 MPNST PDXs. In immunocompetent mice, combination therapy of de novo MPNSTs caused tumor regression, delayed resistant tumor outgrowth, and improved survival relative to monotherapies. Drug-sensitive tumors that regressed contained plasma cells and increased cytotoxic T cells, whereas drug-resistant tumors adopted an immunosuppressive microenvironment with elevated MHC II-low macrophages and increased tumor cell PD-L1 expression. Excitingly, CDK4/6-MEK inhibition sensitized MPNSTs to anti-PD-L1 immune checkpoint blockade (ICB) with some mice showing complete tumor regression. CDK4/6-MEK inhibition induces a novel plasma cell-associated immune response and extended antitumor activity in MPNSTs, which dramatically enhances anti-PD-L1 therapy. These preclinical findings provide strong rationale for clinical translation of CDK4/6-MEK-ICB targeted therapies in MPNST as they may yield sustained antitumor responses and improved patient outcomes.

Repeatedly next-generation sequencing during treatment follow-up of patients with small cell lung cancer.

Somatic alterations in tumors are a frequent occurrence. In small cell lung cancer (SCLC), these include mutations in the tumor suppressors TP53 and retinoblastoma (RB1). We used next generation sequencing (NGS) to study specific genetic variants and compare genetic and clinicopathological features of SCLC with healthy control genome. Ten SCLC patients receiving standard chemotherapy, between 2018 and 2019, from the First Hospital of Jilin University were included in this study. Prior patient treatment, NGS was performed using DNA isolated from blood plasma. New NGS analyses were performed after 2 and 4 treatment cycles. Four patients presented with different metastases at diagnosis. Overall, most genes tested presented missense or frameshift variants. TP53, RB1, CREBBP, FAT1 genes presented gain of stop codons. At the single-gene level, the most frequently altered genes were TP53 (8/10 patients, 80%) and RB1 (4/10 patients, 40%), followed by bromodomain containing 4 (BRD4), CREBBP, FAT1, FMS-like tyrosine kinase 3 (FLT3), KDR, poly ADP-ribose polymerase (PARP1), PIK3R2, ROS1, and splicing factor 3b subunit 1 (SF3B1) (2/10 patients, 20%). We identified 5 genes, which have not been previously reported to bear mutations in the context of SCLC. These genes include BRD4, PARP1, FLT3, KDR, and SF3B1. We observed that among the studied individuals, patients with a high number of genetic events, and in which such mutations were not eradicated after treatment, showed a worse prognosis. There has not yet been given enough attention to the above-mentioned genes in SCLC, which will have great clinical prospects for treatment.