Pre-Clinical Studies of a Novel Bispecific Fusion Protein Targeting C3b and VEGF in Neovascular and Nonexudative AMD Models.

INTRODUCTION: KNP-301 is a bi-specific fragment crystallizable region (Fc) fusion protein, which inhibits both C3b and vascular endothelial growth factor (VEGF) simultaneously for patients with late-stage age-related macular degeneration (AMD). The present study evaluated in vitro potency, in vivo efficacy, intravitreal pharmacokinetics (IVT PK), and injectability of KNP-301. METHODS: C3b and VEGF binding of KNP-301 were assessed by surface plasmon resonance (SPR) and enzyme-linked immunosorbent assay (ELISA), and cellular bioassays. A laser-induced choroidal neovascularization (CNV) model and a sodium iodate-induced nonexudative AMD model were used to test the in vivo efficacy of mouse surrogate of KNP-301. Utilizing fluorescein angiography (FA) and spectral-domain optical coherence tomography (SD-OCT) scans, the reduction in disease lesions were analyzed in a CNV mouse model. In the nonexudative AMD mouse model, outer nuclear layer (ONL) was assessed by immunofluorescence staining. Lastly, intravitreal pharmacokinetic study was conducted with New Zealand white rabbits via IVT administration of KNP-301 and injectability of KNP-301 was examined by a viscosity test at high concentrations. RESULTS: KNP-301 bound C3b selectively, which resulted in a blockade of the alternative pathway, not the classical pathway. KNP-301 also acted as a VEGF trap, impeding VEGF-mediate signaling. Our dual-blockade strategy was effective in both neovascular and nonexudative AMD models. Moreover, KNP-301 had an advantage of potentially less frequent dosing due to the long half-life in the intravitreal chamber. Our viscosity assessment confirmed that KNP-301 meets the criteria of the IVT injection. CONCLUSIONS: Unlike current therapies, KNP-301 is expected to cover patients with late-stage AMD of both neovascular and nonexudative AMD, and its long-term PK profile at the intravitreal chamber would allow convenience in the dosing interval of patients.

Thinking (Metastasis) outside the (Primary Tumor) Box.

The metastasis of tumor cells into vital organs is a major cause of death from diverse types of malignancies [...].

Distinct shared and compartment-enriched oncogenic networks drive primary versus metastatic breast cancer.

Metastatic breast-cancer is a major cause of death in women worldwide, yet the relationship between oncogenic drivers that promote metastatic versus primary cancer is still contentious. To elucidate this relationship in treatment-naive animals, we hereby describe mammary-specific transposon-mutagenesis screens in female mice together with loss-of-function Rb, which is frequently inactivated in breast-cancer. We report gene-centric common insertion-sites (gCIS) that are enriched in primary-tumors, in metastases or shared by both compartments. Shared-gCIS comprise a major MET-RAS network, whereas metastasis-gCIS form three additional hubs: Rho-signaling, Ubiquitination and RNA-processing. Pathway analysis of four clinical cohorts with paired primary-tumors and metastases reveals similar organization in human breast-cancer with subtype-specific shared-drivers (e.g. RB1-loss, TP53-loss, high MET, RAS, ER), primary-enriched (EGFR, TGFß and STAT3) and metastasis-enriched (RHO, PI3K) oncogenic signaling. Inhibitors of RB1-deficiency or MET plus RHO-signaling cooperate to block cell migration and drive tumor cell-death. Thus, targeting shared- and metastasis- but not primary-enriched derivers offers a rational avenue to prevent metastatic breast-cancer.

A temporal in vivo catalog of chromatin accessibility and expression profiles in pineoblastoma reveals a prevalent role for repressor elements.

Pediatric pineoblastomas (PBs) are rare and aggressive tumors of grade IV histology. Although some oncogenic drivers are characterized, including germline mutations in RB1 and DICER1, the role of epigenetic deregulation and cis-regulatory regions in PB pathogenesis and progression is largely unknown. Here, we generated genome-wide gene expression, chromatin accessibility, and H3K27ac profiles covering key time points of PB initiation and progression from pineal tissues of a mouse model of CCND1-driven PB. We identified PB-specific enhancers and super-enhancers, and found that in some cases, the accessible genome dynamics precede transcriptomic changes, a characteristic that is underexplored in tumor progression. During progression of PB, newly acquired open chromatin regions lacking H3K27ac signal become enriched for repressive state elements and harbor motifs of repressor transcription factors like HINFP, GLI2, and YY1. Copy number variant analysis identified deletion events specific to the tumorigenic stage, affecting, among others, the histone gene cluster and Gas1, the growth arrest specific gene. Gene set enrichment analysis and gene expression signatures positioned the model used here close to human PB samples, showing the potential of our findings for exploring new avenues in PB management and therapy. Overall, this study reports the first temporal and in vivo cis-regulatory, expression, and accessibility maps in PB.

A racemosin B derivative, C25, suppresses breast cancer growth via lysosomal membrane permeabilization and inhibition of autophagic flux.

Breast cancer is the most common malignancy among women worldwide. As conventional therapies are only partially successful in eradicating breast cancer, the development of novel strategies is a top priority. We previously showed that C25, a new racemosin B derivative, exerts its anti-cancer activity through inhibition of autophagy, but the underlying mechanism remained unknown. Here we show that C25 inhibits the growth of diverse breast cancer cell subtypes and effectively suppresses tumor progression in a xenotransplantation model of triple negative breast cancer. C25 acts as a lysosomotropic agent to induce lysosomal membrane permeabilization and inhibit autophagic flux, resulting in cathepsin release and cell death. In accordance, RNA sequencing and gene set enrichment analysis revealed that C25 induces pathways consistent with autophagy inhibition, cell cycle arrest and senescence. Interestingly, knockdown of TFEB or SQSTM1 reduced cell death induced by C25 treatment. Finally, we show that C25 synergizes with the chemo-therapeutics etoposide and paclitaxel to further limit breast cancer cell growth. Thus, C25 alone or in combination with other anti-neoplastic agents offers a novel therapeutic strategy for aggressive forms of breast cancer and possibly other malignancies.

Hypophosphorylated pRb knock-in mice exhibit hallmarks of aging and vitamin C-preventable diabetes.

Despite extensive analysis of pRB phosphorylation in vitro, how this modification influences development and homeostasis in vivo is unclear. Here, we show that homozygous Rb∆K4 and Rb∆K7 knock-in mice, in which either four or all seven phosphorylation sites in the C-terminal region of pRb, respectively, have been abolished by Ser/Thr-to-Ala substitutions, undergo normal embryogenesis and early development, notwithstanding suppressed phosphorylation of additional upstream sites. Whereas Rb∆K4 mice exhibit telomere attrition but no other abnormalities, Rb∆K7 mice are smaller and display additional hallmarks of premature aging including infertility, kyphosis, and diabetes, indicating an accumulative effect of blocking pRb phosphorylation. Diabetes in Rb∆K7 mice is insulin-sensitive and associated with failure of quiescent pancreatic ß-cells to re-enter the cell cycle in response to mitogens, resulting in induction of DNA damage response (DDR), senescence-associated secretory phenotype (SASP), and reduced pancreatic islet mass and circulating insulin level. Pre-treatment with the epigenetic regulator vitamin C reduces DDR, increases cell cycle re-entry, improves islet morphology, and attenuates diabetes. These results have direct implications for cell cycle regulation, CDK-inhibitor therapeutics, diabetes, and longevity.

Methylation data of mouse Rb-deficient pineoblastoma.

Methylation profiling is widely used to study tumor biology and perform cluster analysis, particularly in brain cancer research where tissue biopsies are scarce. We have recently reported on the development of novel mouse models for germ line mutations in pineoblastoma (Nature Communications, 2020). Here, we present unpublished methylation profiling of 8 Rb-deleted/p53-deleted pineoblastoma from our mouse model as well as 3 normal cerebellum tissues as control. The primary dataset can be accessed via SRA (PRJNA638504). These methylation data can be used to perform inter- and intra-species comparisons with other brain cancers as well as with specific subtypes of pineoblastoma, and to investigate potential epigenetic mechanisms and pathways underlying Rb-deficient pineoblastoma-genesis..

Modeling germline mutations in pineoblastoma uncovers lysosome disruption-based therapy.

Pineoblastoma is a rare pediatric cancer induced by germline mutations in the tumor suppressors RB1 or DICER1. Presence of leptomeningeal metastases is indicative of poor prognosis. Here we report that inactivation of Rb plus p53 via a WAP-Cre transgene, commonly used to target the mammary gland during pregnancy, induces metastatic pineoblastoma resembling the human disease with 100% penetrance. A stabilizing mutation rather than deletion of p53 accelerates metastatic dissemination. Deletion of Dicer1 plus p53 via WAP-Cre also predisposes to pineoblastoma, albeit with lower penetrance. In silico analysis predicts tricyclic antidepressants such as nortriptyline as potential therapeutics for both pineoblastoma models. Nortriptyline disrupts the lysosome, leading to accumulation of non-functional autophagosome, cathepsin B release and pineoblastoma cell death. Nortriptyline further synergizes with the antineoplastic drug gemcitabine to effectively suppress pineoblastoma in our preclinical models, offering new modality for this lethal childhood malignancy.

Cdh1 and Pik3ca Mutations Cooperate to Induce Immune-Related Invasive Lobular Carcinoma of the Breast.

CDH1 and PIK3CA are the two most frequently mutated genes in invasive lobular carcinoma (ILC) of the breast. Transcription profiling has identified molecular subtypes for ILC, one of which, immune-related (IR), is associated with gene expression linked to lymphocyte and macrophage infiltration. Here, we report that deletion of Cdh1, together with activation of Pik3ca in mammary epithelium of genetically modified mice, leads to formation of IR-ILC-like tumors with immune cell infiltration, as well as gene expression linked to T-regulatory (Treg) cell signaling and activation of targetable immune checkpoint pathways. Interestingly, these tumors show enhanced Rac1- and Yap-dependent transcription and signaling, as well as sensitivity to PI3K, Rac1, and Yap inhibitors in culture. Finally, high-dimensional immunophenotyping in control mouse mammary gland and IR-ILC tumors by mass cytometry shows dramatic alterations in myeloid and lymphoid populations associated with immune suppression and exhaustion, highlighting the potential for therapeutic intervention via immune checkpoint regulators.

CDC25 as a common therapeutic target for triple-negative breast cancer - the challenges ahead.

The dual phosphatase CDC25 has recently been identified as a target for diverse triple-negative breast cancers including RB1/PTEN/P53-deficient tumors. Moreover, CDC25 inhibitors effectively synergize with PI3K inhibitors to suppress tumor growth. We discuss these findings and the challenges that lie ahead in bringing CDC25 inhibitors to the clinic.

Mitochondrial OXPHOS Induced by RB1 Deficiency in Breast Cancer: Implications for Anabolic Metabolism, Stemness, and Metastasis.

A switch from catabolic to anabolic metabolism, a major hallmark of cancer, enables rapid cell duplication, and is driven by multiple oncogenic alterations, including PIK3CA mutation, MYC amplification, and TP53 loss. However, tumor growth requires active mitochondrial function and oxidative phosphorylation (OXPHOS). Recently, loss of the retinoblastoma (RB1) tumor suppressor in breast cancer was shown to induce mitochondrial protein translation (MPT) and OXPHOS. Here, we discuss how increased OXPHOS can enhance anabolic metabolism and cell proliferation, as well as cancer stemness and metastasis. Mitochondrial STAT3, FER/FER-T, and CHCHD2 are also implicated in OXPHOS. We propose that RB1 loss represents a prototypic oncogenic alteration that promotes OXPHOS, that aggressive tumors acquire lethal combinations of oncogenes and tumor suppressors that stimulate anabolism versus OXPHOS, and that targeting both metabolic pathways would be therapeutic.

RB1 deficiency in triple-negative breast cancer induces mitochondrial protein translation.

Triple-negative breast cancer (TNBC) includes basal-like and claudin-low subtypes for which no specific treatment is currently available. Although the retinoblastoma tumor-suppressor gene (RB1) is frequently lost together with TP53 in TNBC, it is not directly targetable. There is thus great interest in identifying vulnerabilities downstream of RB1 that can be therapeutically exploited. Here, we determined that combined inactivation of murine Rb and p53 in diverse mammary epithelial cells induced claudin-low-like TNBC with Met, Birc2/3-Mmp13-Yap1, and Pvt1-Myc amplifications. Gene set enrichment analysis revealed that Rb/p53-deficient tumors showed elevated expression of the mitochondrial protein translation (MPT) gene pathway relative to tumors harboring p53 deletion alone. Accordingly, bioinformatic, functional, and biochemical analyses showed that RB1-E2F complexes bind to MPT gene promoters to regulate transcription and control MPT. Additionally, a screen of US Food and Drug Administration-approved (FDA-approved) drugs identified the MPT antagonist tigecycline (TIG) as a potent inhibitor of Rb/p53-deficient tumor cell proliferation. TIG preferentially suppressed RB1-deficient TNBC cell proliferation, targeted both the bulk and cancer stem cell fraction, and strongly attenuated xenograft growth. It also cooperated with sulfasalazine, an FDA-approved inhibitor of cystine xCT antiporter, in culture and xenograft assays. Our results suggest that RB1 deficiency promotes cancer cell proliferation in part by enhancing mitochondrial function and identify TIG as a clinically approved drug for RB1-deficient TNBC.

shRNA kinome screen identifies TBK1 as a therapeutic target for HER2+ breast cancer.

HER2(+) breast cancer is currently treated with chemotherapy plus anti-HER2 inhibitors. Many patients do not respond or relapse with aggressive metastatic disease. Therefore, there is an urgent need for new therapeutics that can target HER2(+) breast cancer and potentiate the effect of anti-HER2 inhibitors, in particular those that can target tumor-initiating cells (TIC). Here, we show that MMTV-Her2/Neu mammary tumor cells cultured as nonadherent spheres or as adherent monolayer cells select for stabilizing mutations in p53 that "immortalize" the cultures and that, after serial passages, sphere conditions maintain TICs, whereas monolayer cells gradually lose these tumorigenic cells. Using tumorsphere formation as surrogate for TICs, we screened p53-mutant Her2/Neu(+) tumorsphere versus monolayer cells with a lentivirus short hairpin RNA kinome library. We identified kinases such as the mitogen-activated protein kinase and the TGFßR protein family, previously implicated in HER2(+) breast cancer, as well as autophagy factor ATG1/ULK1 and the noncanonical IκB kinase (IKK), TANK-binding kinase 1 (TBK1), which have not been previously linked to HER2(+) breast cancer. Knockdown of TBK1 or pharmacologic inhibition of TBK1 and the related protein, IKKε, suppressed growth of both mouse and human HER2(+) breast cancer cells. TBK1/IKKε inhibition promoted cellular senescence by suppressing p65-NF-κB and inducing p16(Ink4a). In addition, TBK1/IKKε inhibition cooperated with lapatinib, a HER2/EGFR1-targeted drug, to accelerate apoptosis and kill HER2(+) breast cancer cells both in culture and in xenografts. Our results suggest that patients with HER2(+) breast cancer may benefit from anti-TBK1/IKKε plus anti-HER2 combination therapies and establish conditions that can be used to screen for additional TIC-specific inhibitors of HER2(+) breast cancer.

Targeting HER2(+) breast cancer: the TBK1/IKKε axis.

HER2(+) breast cancer (BC) is a highly aggressive subtype, affecting ~20% of BC patients. Current treatments include adjuvant or neoadjuvant chemotherapy plus anti-HER2 agents such as trastuzumab, a monoclonal antibody directed against HER2. Despite improvement in disease free survival, most patients eventually succumb to metastatic disease, which is largely incurable. Consequently, there is an urgent need to identify novel drugs that can efficiently kill HER2(+) BC and/or potentiate the effect of existing anti-HER2 therapies. We performed a lenti-viral shRNA kinome screen on non-adherent mouse Her2/Neu tumorspheres and identified TBK1, a non-canonical IκB kinase (IKK), as the most potent target [1]. TBK1 knock-down, or treatment with TBK1-II, a drug that efficiently inhibits TBK1 and its close relative IKKε (IKBKE), suppressed growth of human HER2(+) BC cells and induced cellular senescence. Senescence was associated with inhibition of phosphorylated/active p65-NFkB and induction of the cell cycle inhibitor, p16(ink4a). In addition, TBK1-II cooperated with lapatinib, a EGFR/HER2 inhibitor, to accelerate apoptosis in vitro and suppress tumor growth in a xenograft model of HER2(+) BC. Thus, TBK1/IKKε inhibitors may improve treatment of HER2(+) BC in cooperation with anti-HER2 therapy.

RB1 and p53 at the crossroad of EMT and triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous disease that includes Basal-like and Claudin-low tumors. The Claudin-low tumors are enriched for features associated with epithelial-to-mesenchymal transition (EMT) and possibly for tumor initiating cells. Primary TNBCs respond relatively well to conventional chemotherapy; however, metastatic disease is virtually incurable. Thus, there is a great interest in identifying specific therapeutic targets for TNBC. The tumor suppressor RB1 is frequently lost in Basal-like breast cancer. To test for a causative role of RB1 gene loss in BC and for its effect on specific subtypes, we deleted mouse Rb in mammary stem/bipotent progenitor cells. This led to diverse mammary tumors including TNBC, with a subset of the latter containing p53 mutations and exhibiting features of Basal-like BC or EMT. Combined mutation of Rb and p53 in mammary stem/bipotent progenitors induced EMT type tumors. Here, we review our findings and those of others, which connect Rb and p53 to EMT in TNBC. Furthermore, we discuss how by understanding this circuit and its vulnerabilities, we may identify novel therapy for TNBC.