Retinoblastoma Tumor Suppressor Research Articles

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.

Rbf/E2F1 control growth and endoreplication via steroid-independent Ecdysone Receptor signalling in Drosophila prostate-like secondary cells.

In prostate cancer, loss of the tumour suppressor gene, Retinoblastoma (Rb), and consequent activation of transcription factor E2F1 typically occurs at a late-stage of tumour progression. It appears to regulate a switch to an androgen-independent form of cancer, castration-resistant prostate cancer (CRPC), which frequently still requires androgen receptor (AR) signalling. We have previously shown that upon mating, binucleate secondary cells (SCs) of the Drosophila melanogaster male accessory gland (AG), which share some similarities with prostate epithelial cells, switch their growth regulation from a steroid-dependent to a steroid-independent form of Ecdysone Receptor (EcR) control. This physiological change induces genome endoreplication and allows SCs to rapidly replenish their secretory compartments, even when ecdysone levels are low because the male has not previously been exposed to females. Here, we test whether the Drosophila Rb homologue, Rbf, and E2F1 regulate this switch. Surprisingly, we find that excess Rbf activity reversibly suppresses binucleation in adult SCs. We also demonstrate that Rbf, E2F1 and the cell cycle regulators, Cyclin D (CycD) and Cyclin E (CycE), are key regulators of mating-dependent SC endoreplication, as well as SC growth in both virgin and mated males. Importantly, we show that the CycD/Rbf/E2F1 axis requires the EcR, but not ecdysone, to trigger CycE-dependent endoreplication and endoreplication-associated growth in SCs, mirroring changes seen in CRPC. Furthermore, Bone Morphogenetic Protein (BMP) signalling, mediated by the BMP ligand Decapentaplegic (Dpp), intersects with CycD/Rbf/E2F1 signalling to drive endoreplication in these fly cells. Overall, our work reveals a signalling switch, which permits rapid growth of SCs and increased secretion after mating, independently of previous exposure to females. The changes observed share mechanistic parallels with the pathological switch to hormone-independent AR signalling seen in CRPC, suggesting that the latter may reflect the dysregulation of a currently unidentified physiological process.

Methyltransferase-independent function of enhancer of zeste homologue 2 maintains tumorigenicity induced by human oncogenic papillomavirus and polyomavirus

Merkel cell polyomavirus (MCV) and high-risk human papillomavirus (HPV) are human tumor viruses that cause Merkel cell carcinoma (MCC) and oropharyngeal squamous cell carcinoma (OSCC), respectively. HPV E7 and MCV large T (LT) oncoproteins target the retinoblastoma tumor suppressor protein (pRb) through the conserved LxCxE motif. We identified enhancer of zeste homolog 2 (EZH2) as a common host oncoprotein activated by both viral oncoproteins through the pRb binding motif. EZH2 is a catalytic subunit of the polycomb 2 (PRC2) complex that trimethylates histone H3 at lysine 27 (H3K27me3). In MCC tissues EZH2 was highly expressed, irrespective of MCV status. Loss-of-function studies revealed that viral HPV E6/E7 and T antigen expression are required for Ezh2 mRNA expression and that EZH2 is essential for HPV(+)OSCC and MCV(+)MCC cell growth. Furthermore, EZH2 protein degraders reduced cell viability efficiently and rapidly in HPV(+)OSCC and MCV(+)MCC cells, whereas EZH2 histone methyltransferase inhibitors did not affect cell proliferation or viability within the same treatment period. These results suggest that a methyltransferase-independent function of EZH2 contributes to tumorigenesis downstream of two viral oncoproteins, and that direct targeting of EZH2 protein expression could be a promising strategy for the inhibition of tumor growth in HPV(+)OSCC and MCV(+)MCC patients.

CDK4/6 inhibitors and the pRB-E2F1 axis suppress PVR and PD-L1 expression in triple-negative breast cancer

Immune-checkpoint (IC) modulators like the poliovirus receptor (PVR) and programmed death ligand 1 (PD-L1) attenuate innate and adaptive immune responses and are potential therapeutic targets for diverse malignancies, including triple-negative breast cancer (TNBC). The retinoblastoma tumor suppressor, pRB, controls cell growth through E2F1-3 transcription factors, and its inactivation drives metastatic cancer, yet its effect on IC modulators is contentious. Here, we show that RB-loss and high E2F1/E2F2 signatures correlate with expression of PVR, CD274 (PD-L1 gene) and other IC modulators and that pRB represses whereas RB depletion and E2F1 induce PVR and CD274 in TNBC cells. Accordingly, the CDK4/6 inhibitor, palbociclib, suppresses both PVR and PD-L1 expression. Palbociclib also counteracts the effect of CDK4 on SPOP, leading to its depletion, but the overall effect of palbociclib is a net reduction in PD-L1 level. Hydrochloric acid, commonly used to solubilize palbociclib, counteracts its effect and induces PD-L1 expression. Remarkably, lactic acid, a by-product of glycolysis, also induces PD-L1 as well as PVR. Our results suggest a model in which CDK4/6 regulates PD-L1 turnover by promoting its transcription via pRB-E2F1 and degradation via SPOP and that the CDK4/6-pRB-E2F pathway couples cell proliferation with the induction of multiple innate and adaptive immunomodulators, with direct implications for cancer progression, anti-CDK4/6- and IC-therapies.

Seeing is believing: the impact of RB on nuclear organization

ABSTRACT The retinoblastoma tumor suppressor (RB) prevents G1 to S cell cycle transition by inhibiting E2F activity. This function requires that RB remains un- or underphosphorylated (the so-called active forms of RB). Recently, we showed that active forms of RB cause widespread changes in nuclear architecture that are visible under a microscope. These phenotypes did not correlate with cell cycle arrest or repression of the E2F transcriptional program, but appeared later, and were associated with the appearance of autophagy or in IMR-90 cells with senescence markers. In this perspective, we describe the relative timing of these RB-induced events and discuss the mechanisms that may underlie RB-induced chromatin dispersion. We consider the relationship between RB-induced dispersion, autophagy, and senescence and the potential connection between dispersion and cell cycle exit.

Identification of Potential p38γ Inhibitors via In Silico Screening, In Vitro Bioassay and Molecular Dynamics Simulation Studies.

Protein kinase p38γ is an attractive target against cancer because it plays a pivotal role in cancer cell proliferation by phosphorylating the retinoblastoma tumour suppressor protein. Therefore, inhibition of p38γ with active small molecules represents an attractive alternative for developing anti-cancer drugs. In this work, we present a rigorous and systematic virtual screening framework to identify potential p38γ inhibitors against cancer. We combined the use of machine learning-based quantitative structure activity relationship modelling with conventional computer-aided drug discovery techniques, namely molecular docking and ligand-based methods, to identify potential p38γ inhibitors. The hit compounds were filtered using negative design techniques and then assessed for their binding stability with p38γ through molecular dynamics simulations. To this end, we identified a promising compound that inhibits p38γ activity at nanomolar concentrations and hepatocellular carcinoma cell growth in vitro in the low micromolar range. This hit compound could serve as a potential scaffold for further development of a potent p38γ inhibitor against cancer.

Abstract 5989: Targeting RRM2 & cell cycle for breast cancer treatment

Abstract Endocrine treatment is an effective first-line therapy for targeting ER positive, HER2 negative breast cancer. But success is limited by development of acquired resistance due to long-term therapy. Cyclin D1 and cyclin dependent kinase 4/6 (CDK4/6) complex causes phosphorylation and subsequent inactivation of retinoblastoma (Rb) tumor suppressor protein which promotes progression of the cell cycle from G1 to S phase. This observation led the development of the first CDK4/6 inhibitor palbociclib (Ibrance; Pfizer) which induces cell cycle arrest at G1 phase in cancer cells. Owing to intrinsic and acquired drug resistance development, success is limited despite promising clinical outcomes. This situation necessitates the development of potential combination strategies to overcome drug resistance. Ribonucleotide reductase (RR) is a rate limiting enzyme in DNA synthesis consisting of two subunits RRM1 and RRM2. Didox inhibits ribonucleotide reductase subunit 2 (RRM2) which ultimately blocks DNA synthesis. Combination of didox with palbociclib is a potential strategy to target ER positive and ER negative/triple negative breast cancer. Previously, we have shown that didox (DDX) can significantly halt malignant breast cancer cell division in combination with the anthracycline drug doxorubicin by targeting RRM2, mutant p53 and NFkB regulatory proteins. Recently, we confirmed that didox in combination with palbociclib significantly lowers ER positive and ER negative breast cancer and their palbociclib resistant counterparts’ growth compared to no treatment or palbociclib treatment. We also confirmed that ER positive MCF7 and ER negative MDA-MB-468 parental breast cancer cells IC50 of palbociclib drug is lower than their palbociclib resistant counterparts. Here, we are reporting that didox decreases cell cycle proteins alone or in combination with palbociclib in ER positive MCF7 and ER negative MDA-MB-468 parental and palbociclib resistant breast cancer cells. This finding opens a novel approach for targeting both ER positive as well as ER negative breast cancer treatment. We are also reporting that didox treatment modulates cyclin D1 (CCND1) and RRM2 expression in MCF7 and MDA-MB-468 breast cancer along with their palbociclib resistant counterparts. Additionally, we observed that didox alone or in combination with palbociclib alters the cell cycle of MCF7 and MDA-MB-468 parental and palbociclib resistant breast cancer cells. Our data presents a novel and promising approach for the treatment of ER positive and ER negative breast cancer that involves inhibition of RRM2 and cell cycle that merits further clinical investigation in animal and human models. Citation Format: Nahid Sultana, Howard L. Elford, Jesika S. Faridi. Targeting RRM2 & cell cycle for breast cancer treatment. [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 5989.

Abstract 1564: Novel combination of TRIP13 and Aurora kinase A inhibition demonstrated extensive DNA damage and immunogenic cell death in RB-deficient cancers

Abstract Many lethal cancer types have inactivation of the retinoblastoma (Rb) tumor suppressor pathway including cancers caused by human papillomavirus (HPV), which causes >5% of all cancers worldwide. There are no therapies that uniquely target Rb-deficient cancers. Biomarker-selected, molecular targeted therapy for Rb-deficient cancers represents both an unmet need and a translational knowledge gap. To address this gap, we recently published data that support a model in which Rb-deficient cancers, having high levels of Mad2, survive owing to TRIP13-mediated inhibition of Mad2 and Aurora A-mediated mitotic progression. The combination of Aurora A inhibition (alisertib) plus TRIP13 depletion caused extensive apoptosis in Rb-deficient, but not in Rb-proficient, cancer cells. Both Aurora A and TRIP13 are involved in the cell’s response to DNA damage and apoptosis that can lead to immunogenic cell death (ICD), prompting us to investigate the mechanism underlying the synthetic lethality of TRIP13 and Aurora A in Rb-deficient cancer cells. We transfected two HPV+ cell lines with TRIP13 or control siRNA and incubated with 100nM alisertib. We also generated two stable HPV+ cell lines with TRIP13KO. In both these systems, TRIP13 KD or KO alone did not cause apoptosis. Incubation with alisertib resulted in significant cell death, that validated our previous findings. We then tested the effect of the combination on DNA damage. Alisertib caused robust increases in γH2AX, pTIF1β (KAP1, TRIM28), and DNA PKcs, measured using immunoblotting, in both TRIP13 KD and KO HPV+ cell lines. We also observed enhanced γH2AX staining using confocal microscopy. ICD leads to activation of immune cells by the dying cancer cells in the tumor microenvironment involving the release of antigenic fragments that are engulfed, processed and presented by antigen presenting cells for the induction of antigen-specific T-cell responses. Studies have shown DNA damage stimulates the cGAS/STING pathway leading to ICD. In our system, we saw that TRIP13KO and KD in HPV+ cells treated with alisertib, resulted in marked activation of the cGAS/STING pathway and release of cytochrome C and IL1α in the cell supernatant, which are markers of pyroptosis. Additionally, we also observed gasdermin-E cleavage. The combination further led to increased cancer cell secretion of GM-CSF, IL6, and IL18 that can activate macrophages, NK cells and T-cells. The selective effect of the combination in Rb-deficient cancer cells allowed us to significantly reduce the concentration of alisertib. By sparing normal cells that express Rb, this combination can potentially reduce treatment toxicity in patients with Rb-deficient cancers. The ICD induced by this combination may lead to host T-cell engagement, thereby further enhancing tumor elimination. This work can shape future clinical trials that can change current treatment regime. Citation Format: Soma Ghosh, Tuhina Mazumdar, Lacin Yapindi, Pragya Sinha, Jagannadha Sastry, Faye M. Johnson. Novel combination of TRIP13 and Aurora kinase A inhibition demonstrated extensive DNA damage and immunogenic cell death in RB-deficient cancers [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 1564.

CDK6 activity in a recurring convergent kinase network motif.

In humans, more than 500 kinases phosphorylate ~15% of all proteins in an emerging phosphorylation network. Convergent local interaction motifs, in which ≥two kinases phosphorylate the same substrate, underlie feedback loops and signal amplification events but have not been systematically analyzed. Here, we first report a network-wide computational analysis of convergent kinase-substrate relationships (cKSRs). In experimentally validated phosphorylation sites, we find that cKSRs are common and involve >80% of all human kinases and >24% of all substrates. We show that cKSRs occur over a wide range of stoichiometries, in many instances harnessing co-expressed kinases from family subgroups. We then experimentally demonstrate for the prototypical convergent CDK4/6 kinase pair how multiple inputs phosphorylate the tumor suppressor retinoblastoma protein (RB) and thereby hamper in situ analysis of the individual kinases. We hypothesize that overexpression of one kinase combined with a CDK4/6 inhibitor can dissect convergence. In breast cancer cells expressing high levels of CDK4, we confirm this hypothesis and develop a high-throughput compatible assay that quantifies genetically modified CDK6 variants and inhibitors. Collectively, our work reveals the occurrence, topology, and experimental dissection of convergent interactions toward a deeper understanding of kinase networks and functions.

IL-6 evoked biochemical changes in prostate cancer cells.

The pro-inflammatory cytokine IL-6 has been associated with the progression of PCa to a castration-resistant phenotype. In this work, we characterized the biochemical changes evoked by IL-6 in three different models of PCa cells, including LNCaP, C4-2, and PC3. The effect of IL-6 on PCa cells was compared with the effect obtained by co-stimulation with the cAMP-inducing agent forskolin (FSK). Stimulation of LNCaP cells with IL-6 or IL-6+FSK evoked increased expression of the neuroendocrine marker tubulin IIIβ and Cav3.2T-type Ca2+ channel subunit. PC3 cells, representing a more advanced state of PCa, had high levels of tubulin IIIβ expression without any further changes observed by treatment with IL-6 or IL-6+FSK. Elevated expression of the glucocorticoid receptor was observed in PC3, but not in LNCaP or C4-2 cells. Glucocorticoid receptor expression was not regulated by IL-6 stimulation of LNCaP or C4-2 cells. IL-6 acting alone or together with FSK evoked a significant reduction in the expression of the transcription factor REST and retinoblastoma tumor suppressor protein Rb1. In LNCaP cells, IL-6 acting alone or together with FSK had no effect on the expression of several biological markers of advanced PCa, including Aurora kinase A, valosin-containing protein, calcium-sensing receptor, calreticulin, S100A protein, and Protein S. In PC3 cells, co-treatment with IL-6+FSK evoked increased expression of REST and S100A proteins, as well as a reduction in Protein S levels. These findings reveal a complex pattern of biochemical changes in PCa cells under the influence of IL-6.

Discovery of cancer-preventive juices reactivating RB functions.

Recent advances have been achieved in the genetic diagnosis and therapies against malignancies due to a better understanding of the molecular mechanisms underlying carcinogenesis. Since active preventive methods are currently insufficient, the further development of appropriate preventive strategies is desired. We searched for drinks that reactivate the functions of tumor-suppressor retinoblastoma gene (RB) products and exert anti-inflammatory and antioxidant effects. We also examined whether lactic acid bacteria increased the production of the cancer-specific anti-tumor cytokine, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), in human, and examined whether the RB-reactivating drinks with lactic acid bacteria decreased azoxymethane-induced rat colon aberrant crypt foci (ACF) and aberrant crypts (ACs) in vivo. Kakadu plum juice and pomegranate juice reactivated RB functions, which inhibited the growth of human colon cancer LIM1215 cells by G1 phase arrest. These juices also exerted anti-inflammatory and antioxidant effects. Lactiplantibacillus (L.) pentosus S-PT84 was administered to human volunteers and increased the production of TRAIL. In an in vivo study, Kakadu plum juice with or without pomegranate juice and S-PT84 significantly decreased azoxymethane-induced rat colon ACF and ACs. RB is one of the most important molecules suppressing carcinogenesis, and to the best of our knowledge, this is the first study to demonstrate that natural drinks reactivated the functions of RB. As expected, Kakadu plum juice and pomegranate juice suppressed the growth of LIM1215 cells by reactivating the functions of RB, and Kakadu plum juice with or without pomegranate juice and S-PT84 inhibited rat colon ACF and ACs. Therefore, this mixed juice has potential as a novel candidate for cancer prevention.

Combined inhibition of ACLY and CDK4/6 reduces cancer cell growth and invasion.

The use of small molecule kinase inhibitors, which target specific enzymes that are overactive in cancer cells, has revolutionized cancer patient treatment. To treat some types of breast cancer, CDK4/6 inhibitors, such as palbociclib, have been developed that target the phosphorylation of the retinoblastoma tumor suppressor gene. Acquired resistance to CDK4/6 inhibitors may be due to activation of the AKT pro‑survival signaling pathway that stimulates several processes, such as growth, metastasis and changes in metabolism that support rapid cell proliferation. The aim of the present study was to investigate whether targeting ATP citrate lyase (ACLY), a downstream target of AKT, may combine with CDK4/6 inhibition to inhibit tumorigenesis. The present study determined that ACLY is activated in breast and pancreatic cancer cells in response to palbociclib treatment and AKT mediates this effect. Inhibition of ACLY using bempedoic acid used in combination with palbociclib reduced cell viability in a panel of breast and pancreatic cancer cell lines. This effect was also observed using breast cancer cells grown in 3D cell culture. Mechanistically, palbociclib inhibited cell proliferation, whereas bempedoic acid stimulated apoptosis. Finally, using Transwell invasion assays and immunoblotting, the present study demonstrated that ACLY inhibition blocked cell invasion, when used alone or in combination with palbociclib. These data may yield useful information that could guide the development of future therapies aimed at the reduction of acquired resistance observed clinically.

The p16 Antagonist Gankyrin Is Overexpressed in Melanocytic Neoplasms

Gankyrin has a household function in essentially all cells by acting as a chaperone in the assembly of the 26S proteasome, but also functions as a tumor-promoting protein by antagonizing the tumor suppressors retinoblastoma protein, p16, and p53. While gankyrin is overexpressed in many neoplasms outside the skin, its expression in normal skin and cutaneous neoplasms has not been reported previously. We studied the expression of gankyrin in archival human formalin-fixed tissues of cutaneous neoplasms by immunohistochemistry with a monoclonal antibody, and found gankyrin to be overexpressed in 3 of 20 squamous cell carcinomas, none of 10 basal cell carcinomas, 13 of 18 melanocytic nevi, and 7 of 10 melanomas, in many cases with a predominantly nuclear location. Normal epidermal melanocytes expressed gankyrin to a lesser extent than neoplastic melanocytes. The overexpression in the in situ stage of squamous cell carcinoma and in melanocytic nevi suggests that gankyrin acts as a tumor-promoting protein in the early stages of the transition from normal to neoplastic cells. The frequent overexpression of gankyrin in melanocytic neoplasms is significant because it antagonizes the tumor suppressor, p16, which is strongly expressed in melanocytic nevi and some melanomas.

The Cyclin-G Associated Kinase (GAK) Is a Critical Dependency for Cell-Cycle Progression in Diffuse Large B-Cell Lymphoma Associated with Deregulation of the Retinoblastoma Tumor Suppressor

Background: Recurrent genomic alterations in specific cancers reveal tumor drivers that inform the development of rational therapeutic approaches. In diffuse large B-cell lymphoma (DLBCL), such oncoprotein-focused drug development has achieved only limited success. Phenotypic screening, less explored in DLBCL drug discovery, is an emerging alternative that can be leveraged especially through the application of machine-learning analyses to raw screening data. Under this paradigm, we reveal strong therapeutic potential in DLBCL of inhibiting the cyclin-G associated kinase (GAK). GAK participates in clathrin-mediated endocytosis and regulates mitotic-spindle alignment during cell division. It has been studied as an antiviral target that interrupts internalization at cell membranes but is minimally explored in cancer therapeutics and is a novel target in DLBCL. Methods and Results: We performed a first-of-its-kind screen of kinase inhibitors in DLBCL followed by machine learning-based analysis using our in-house target identification platform, idTRAX. The screen revealed GAK as a strong target for both activated B-cell (ABC) and germinal center B-cell (GCB) derived cell lines with complete sparing of peripheral-blood mononuclear cells (PBMCs), included as non-malignant controls. We analyzed gene-expression data available for 414 previously untreated DLBCL patients (Lenz cohort) and found tumors with high GAK expression had dramatically worse overall survival (OS) compared to those with low expression (hazard ratio=1.84 (95% conf. int. 1.33-2.53), p=0.0002). Mechanistically, we explored GAK's role in endocytosis but found no change in cell-surface B-cell receptor (BCR) expression after treatment with the GAK tool compound SGC-GAK-1. In addition, there was no evident perturbation of signaling pathways downstream of BCR by immunoblotting. We found instead that GAK inhibition in DLBCL results in profound disruption of progression through mitosis, potently triggering the spindle assembly checkpoint (SAC) and causing accumulation in G2 before onset of apoptosis. We explored whether GAK dependency is induced due to cell cycle deregulation. Analysis of gene-expression data in the Lenz cohort showed a striking correlation between high GAK expression and low expression of RB1, encoding the tumor suppressive master cell-cycle regulator retinoblastoma-associated protein (RB, p<0.0001). The ABC-DLBCL cell lines RIVA and U2932 completely lack RB expression and show high sensitivity and accumulation in G2 in response to SGC-GAK-1 treatment. Immunofluorescent confocal microscopy revealed profound disruption of mitotic spindles and shattering of chromosomes in these cells and, to a lesser extent, in RB-competent lines. SGC-GAK-1 is not optimized for use as a chemical probe in vivo, but target validation through shRNA knockdown in DLBCL patient-derived xenograft (PDX) models is underway and expected at time of presentation. Conclusions: GAK is a novel kinase therapeutic target in DLBCL revealed by our target identification platform, idTRAX. GAK's role in mitotic-spindle alignment represents a critical kinase dependency for DLBCL tumors, in particular those with cell-cycle deregulation due to loss of RB function. Our ongoing efforts to synthesize novel GAK inhibitors and reveal synergistic targets may reveal new therapeutic strategies for translation to clinical trials for DLBCL patients with unmet clinical needs.

Retinoblastoma protein as an intrinsic BRD4 inhibitor modulates small molecule BET inhibitor sensitivity in cancer

Bromodomain and extraterminal (BET) proteins including BRD4 play important roles in oncogenesis and immune inflammation. Here we demonstrate that cancer cells with loss of the retinoblastoma (RB) tumor suppressor became resistant to small molecule bromodomain inhibitors of BET proteins. We find that RB binds to bromodomain-1 (BD1) of BRD4, but binding is impeded by CDK4/6-mediated RB phosphorylation at serine-249/threonine-252 (S249/T252). ChIP-seq analysis shows RB knockdown increases BRD4 occupancy at genomic loci of genes enriched in cancer-related pathways including the GPCR-GNBIL-CREB axis. S249/T252-phosphorylated RB positively correlates with GNBIL protein level in prostate cancer patient samples. BET inhibitor resistance in RB-deficient cells is abolished by co-administration of CREB inhibitor. Our study identifies RB protein as a bona fide intrinsic inhibitor of BRD4 and demonstrates that RB inactivation confers resistance to small molecule BET inhibitors, thereby revealing a regulatory hub that converges RB upstream signaling onto BRD4 functions in diseases such as cancer.

Nanomedicine-Based Gene Delivery for a Truncated Tumor Suppressor RB94 Promotes Lung Cancer Immunity.

Simple SummaryAdvanced therapies have provided substantial clinical benefits in oncology, but low response rates in lung cancer patients remain challenging. Using preclinical models of human lung cancer, we investigated the anti-tumor potency of nanomedicine-based gene delivery for the tumor suppressor RB94 and explored the mechanisms underlying its activity. This nanomedicine (scL-RB94) elicited significant anti-tumor responses reflecting enhanced tumor immunogenicity and reduced immunosuppression. The potential of scL-RB94 to improve outcomes in lung cancer patients is discussed.Because lung cancer remains the most common and lethal of cancers, novel therapeutic approaches are urgently needed. RB94 is a truncated form of retinoblastoma tumor suppressor protein with elevated anti-tumor efficacy. Our investigational nanomedicine (termed scL-RB94) is a tumor-targeted liposomal formulation of a plasmid containing the gene encoding RB94. In this research, we studied anti-tumor and immune modulation activities of scL-RB94 nanocomplex in preclinical models of human non-small cell lung cancer (NSCLC). Systemic treatment with scL-RB94 of mice bearing human NSCLC tumors significantly inhibited tumor growth by lowering proliferation and increasing apoptosis of tumor cells in vivo. scL-RB94 treatment also boosted anti-tumor immune responses by upregulating immune recognition molecules and recruiting innate immune cells such as natural killer (NK) cells. Antibody-mediated depletion of NK cells blunted the anti-tumor activity of scL-RB94, suggesting that NK cells were crucial for the observed anti-tumor activity in these xenograft models. Treatment with scL-RB94 also altered the polarization of tumor-associated macrophages by reducing immune-suppressive M2 macrophages to lower immune suppression in the tumor microenvironment. Collectively, our data suggest that the efficacy of scL-RB94 against NSCLC is due to an induction of tumor cell death as well as enhancement of innate anti-tumor immunity.

PFKFB3 mediates tubular cell death in cisplatin nephrotoxicity by activating CDK4

Nephrotoxicity is a major side effect of cisplatin, a widely used cancer therapy drug. However, the mechanism of cisplatin nephrotoxicity remains unclear and no effective kidney protective strategies are available. Here, we report the induction of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) in both in vitro cell culture and in vivo mouse models of cisplatin nephrotoxicity. Notably, PFKFB3 was mainly induced in the nucleus of kidney tubular cells, suggesting a novel function other than its canonical role in glycolysis. Both pharmacological inhibition and genetic silencing of PFKFB3 led to the suppression of cisplatin-induced apoptosis in cultured renal proximal tubular cells (RPTCs). Moreover, cisplatin-induced kidney injury or nephrotoxicity was ameliorated in renal proximal tubule-specific PFKFB3 knockout mice. Mechanistically, we demonstrated the interaction of PFKFB3 with cyclin-dependent kinase 4 (CDK4) during cisplatin treatment, resulting in CDK4 activation and consequent phosphorylation and inactivation of retinoblastoma tumor suppressor (Rb). Inhibition of CDK4 reduced cisplatin-induced apoptosis in RPTCs and kidney injury in mice. Collectively, this study unveils a novel pathological role of PFKFB3 in cisplatin nephrotoxicity through the activation of the CDK4/Rb pathway, suggesting a new kidney protective strategy for cancer patients by blocking PFKFB3.

BRCA1, BCL2, and the RB Tumor Suppressor have abnormal expressions in lung cancer

The retinoblastoma (RB) gene encodes the retinoblastoma pocket protein, which controls the cell cycle by binding to unphosphorylated E2F transcription factors and inhibiting their activation. The function of BRCA1 and the anti-apoptotic protein Bcl-2 in lung cancer, however, is still debated. Objective:The purpose of this research is to look at the relationship between the cell-cycle proteins BRCA1, BCL2, and RB and lung cancer etiology and progression. Experimental Design: Cases from major hospitals and many private histopathological laboratories between 2018 and 2021 were reviewed for immunohistochemical expression of BRCA1, BCL2, and RB. A total of 60 people (20 healthy people as a control group and 40 patients with lung carcinoma) were reviewed and analyzed for immunohistochemical expression of these genes. Results: In (90.0%) of cases, RB-IHC was overexpressed, according to the data. The BRCA1 overexpression was seen in (95.0 %). Though BCL2 was overexpressed in (92.5%) of the cases. When comparing the healthy and lung cancer groups, there is a highly significant difference at (P&lt;0.01). Conclusion: Overexpression of RB, BRCA1, and BCL2 in lung cancers with little or no regulatory role may suggest mutational events, which act in collaboration with numerous other genetic mutations in these tissues. The study findings indicate that disruption of cell cycle proteins may perform a unique function in lung cancer disease onset and development and suggest that all patients have abnormalities in the BRCA1, BCL2, and RB proteins. have a role in lung carcinomas.