PI3Kß controls immune evasion in PTEN-deficient breast tumours.

Loss of the PTEN tumour suppressor is one of the most common oncogenic drivers across all cancer types1. PTEN is the major negative regulator of PI3K signalling. The PI3Kß isoform has been shown to play an important role in PTEN-deficient tumours, but the mechanisms underlying the importance of PI3Kß activity remain elusive. Here, using a syngeneic genetically engineered mouse model of invasive breast cancer driven by ablation of both Pten and Trp53 (which encodes p53), we show that genetic inactivation of PI3Kß led to a robust anti-tumour immune response that abrogated tumour growth in syngeneic immunocompetent mice, but not in immunodeficient mice. Mechanistically, PI3Kß inactivation in the PTEN-null setting led to reduced STAT3 signalling and increased the expression of immune stimulatory molecules, thereby promoting anti-tumour immune responses. Pharmacological PI3Kß inhibition also elicited anti-tumour immunity and synergized with immunotherapy to inhibit tumour growth. Mice with complete responses to the combined treatment displayed immune memory and rejected tumours upon re-challenge. Our findings demonstrate a molecular mechanism linking PTEN loss and STAT3 activation in cancer and suggest that PI3Kß controls immune escape in PTEN-null tumours, providing a rationale for combining PI3Kß inhibitors with immunotherapy for the treatment of PTEN-deficient breast cancer.

ΔNp63α is a common inhibitory target in oncogenic PI3K/Ras/Her2-induced cell motility and tumor metastasis.

Activation of phosphatidylinositol 3 kinase (PI3K), Ras, and Her2 signaling plays a critical role in cancer development. Hotspot constitutive activating mutations in oncogenes, such as PIK3CA encoding the p110α catalytic subunit or RAS, as well as overexpression of Her2, are frequently found in human tumors and cancers. It has been well established that activation of these oncogenes profoundly promotes tumor metastasis, whereas decreased expression of ΔNp63α, the major protein isoform of the p53-related p63 expressed in epithelial cells, has been associated with cancer metastasis. In this study, we demonstrate that hotspot oncogenic mutations on PIK3CA and RAS, including p110αH1047R, K-RasG12V, and H-RasG12V, as well as activation of Her2, all led to suppression of ΔNp63α expression via Akt-fork-head transcription factor 3a (Akt-FOXO3a) signaling, resulting in increased cell motility and tumor metastasis. Expression of ΔNp63α effectively reversed p110αH1047R-, K-RasG12V-, H-RasG12V-, or Her2-induced cell motility in vitro and tumor metastasis in mouse models. We show that ΔNp63α was a direct FOXO3a transcriptional target and that expression of FOXO3a and ΔNp63α was correlated in human cancer biopsy samples. Together, these results demonstrate that ΔNp63α is a common inhibitory target of oncogenic PI3K, Ras, and Her2, and that ΔNp63α may function as a critical integrator of oncogenic signaling in cancer metastasis.

Metformin Promotes AMP-activated Protein Kinase-independent Suppression of ΔNp63α Protein Expression and Inhibits Cancer Cell Viability.

The blood glucose modifier metformin is used to treat type II diabetes and has also been shown to possess anticancer activities. Recent studies indicate that glucose deprivation can greatly enhance metformin-mediated inhibition of cell viability, but the molecular mechanism involved in this inhibition is unclear. In this study, we report that, under glucose deprivation, metformin inhibited expression of ΔNp63α, a p53 family member involved in cell adhesion pathways, resulting in disruption of cell matrix adhesion and subsequent apoptosis in human squamous carcinoma cells. We further show that metformin promoted ΔNp63α protein instability independent of AMP-activated protein kinase and that WWP1, an E3 ligase of ΔNp63α, was involved in metformin-mediated down-regulation of ΔNp63α levels. In addition, we demonstrate that a combination of metformin and the glycolysis inhibitor 2-deoxy-d-glucose significantly inhibited ΔNp63α expression and also suppressed xenographic tumor growth in vivo In summary, this study reveals a new mechanism for metformin-mediated anticancer activity and suggests a new strategy for treating human squamous cell carcinoma.

Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation.

The p53 tumor suppressor gene plays a critical role in regulation of proliferation, cell death and differentiation. The MDM2 oncoprotein is a major negative regulator for p53 by binding to and targeting p53 for proteasome-mediated degradation. The small molecule inhibitor, nutlin-3, disrupts MDM2-p53 interaction resulting in stabilization and activation of p53 protein. We have previously shown that nutlin-3 activates p53, leading to MDM2 accumulation as concomitant of reduced retinoblastoma (Rb) protein stability. It is well known that Rb is important in muscle development and myoblast differentiation and that rhabdomyosarcoma (RMS), or cancer of the skeletal muscle, typically harbors MDM2 amplification. In this study, we show that nutlin-3 inhibited myoblast proliferation and effectively prevented myoblast differentiation, as evidenced by lack of expression of muscle differentiation markers including myogenin and myosin heavy chain (MyHC), as well as a failure to form multinucleated myotubes, which were associated with dramatic increases in MDM2 expression and decrease in Rb protein levels. These results indicate that nutlin-3 can effectively inhibit muscle cell differentiation.

Selective CDK7 Inhibition Suppresses Cell Cycle Progression and MYC Signaling While Enhancing Apoptosis in Therapy-resistant Estrogen Receptor-positive Breast Cancer.

PURPOSE: Resistance to endocrine therapy (ET) and CDK4/6 inhibitors (CDK4/6i) is a clinical challenge in estrogen receptor (ER)-positive (ER+) breast cancer. Cyclin-dependent kinase 7 (CDK7) is a candidate target in endocrine-resistant ER+ breast cancer models and selective CDK7 inhibitors (CDK7i) are in clinical development for the treatment of ER+ breast cancer. Nonetheless, the precise mechanisms responsible for the activity of CDK7i in ER+ breast cancer remain elusive. Herein, we sought to unravel these mechanisms. EXPERIMENTAL DESIGN: We conducted multi-omic analyses in ER+ breast cancer models in vitro and in vivo, including models with different genetic backgrounds. We also performed genome-wide CRISPR/Cas9 knockout screens to identify potential therapeutic vulnerabilities in CDK4/6i-resistant models. RESULTS: We found that the on-target antitumor effects of CDK7 inhibition in ER+ breast cancer are in part p53 dependent, and involve cell cycle inhibition and suppression of c-Myc. Moreover, CDK7 inhibition exhibited cytotoxic effects, distinctive from the cytostatic nature of ET and CDK4/6i. CDK7 inhibition resulted in suppression of ER phosphorylation at S118; however, long-term CDK7 inhibition resulted in increased ER signaling, supporting the combination of ET with a CDK7i. Finally, genome-wide CRISPR/Cas9 knockout screens identified CDK7 and MYC signaling as putative vulnerabilities in CDK4/6i resistance, and CDK7 inhibition effectively inhibited CDK4/6i-resistant models. CONCLUSIONS: Taken together, these findings support the clinical investigation of selective CDK7 inhibition combined with ET to overcome treatment resistance in ER+ breast cancer. In addition, our study highlights the potential of increased c-Myc activity and intact p53 as predictors of sensitivity to CDK7i-based treatments.

Lysine vitcylation is a novel vitamin C-derived protein modification that enhances STAT1-mediated immune response.

Vitamin C (vitC) is a vital nutrient for health and also used as a therapeutic agent in diseases such as cancer. However, the mechanisms underlying vitC's effects remain elusive. Here we report that vitC directly modifies lysine without enzymes to form vitcyl-lysine, termed "vitcylation", in a dose-, pH-, and sequence-dependent manner across diverse proteins in cells. We further discover that vitC vitcylates K298 site of STAT1, which impairs its interaction with the phosphatase PTPN2, preventing STAT1 Y701 dephosphorylation and leading to increased STAT1-mediated IFN pathway activation in tumor cells. As a result, these cells have increased MHC/HLA class-I expression and activate immune cells in co-cultures. Tumors collected from vitC-treated tumor-bearing mice have enhanced vitcylation, STAT1 phosphorylation and antigen presentation. The identification of vitcylation as a novel PTM and the characterization of its effect in tumor cells opens a new avenue for understanding vitC in cellular processes, disease mechanisms, and therapeutics.

Targeting CDK4 and CDK6 in cancer.

Cyclin-dependent kinase 4 (CDK4) and CDK6 are critical mediators of cellular transition into S phase and are important for the initiation, growth and survival of many cancer types. Pharmacological inhibitors of CDK4/6 have rapidly become a new standard of care for patients with advanced hormone receptor-positive breast cancer. As expected, CDK4/6 inhibitors arrest sensitive tumour cells in the G1 phase of the cell cycle. However, the effects of CDK4/6 inhibition are far more wide-reaching. New insights into their mechanisms of action have triggered identification of new therapeutic opportunities, including the development of novel combination regimens, expanded application to a broader range of cancers and use as supportive care to ameliorate the toxic effects of other therapies. Exploring these new opportunities in the clinic is an urgent priority, which in many cases has not been adequately addressed. Here, we provide a framework for conceptualizing the activity of CDK4/6 inhibitors in cancer and explain how this framework might shape the future clinical development of these agents. We also discuss the biological underpinnings of CDK4/6 inhibitor resistance, an increasingly common challenge in clinical oncology.

Sarm1 activation produces cADPR to increase intra-axonal Ca++ and promote axon degeneration in PIPN.

Cancer patients frequently develop chemotherapy-induced peripheral neuropathy (CIPN), a painful and long-lasting disorder with profound somatosensory deficits. There are no effective therapies to prevent or treat this disorder. Pathologically, CIPN is characterized by a "dying-back" axonopathy that begins at intra-epidermal nerve terminals of sensory neurons and progresses in a retrograde fashion. Calcium dysregulation constitutes a critical event in CIPN, but it is not known how chemotherapies such as paclitaxel alter intra-axonal calcium and cause degeneration. Here, we demonstrate that paclitaxel triggers Sarm1-dependent cADPR production in distal axons, promoting intra-axonal calcium flux from both intracellular and extracellular calcium stores. Genetic or pharmacologic antagonists of cADPR signaling prevent paclitaxel-induced axon degeneration and allodynia symptoms, without mitigating the anti-neoplastic efficacy of paclitaxel. Our data demonstrate that cADPR is a calcium-modulating factor that promotes paclitaxel-induced axon degeneration and suggest that targeting cADPR signaling provides a potential therapeutic approach for treating paclitaxel-induced peripheral neuropathy (PIPN).

Temporal and spatial topography of cell proliferation in cancer.

Proliferation is a fundamental trait of cancer cells, but its properties and spatial organization in tumours are poorly characterized. Here we use highly multiplexed tissue imaging to perform single-cell quantification of cell cycle regulators and then develop robust, multivariate, proliferation metrics. Across diverse cancers, proliferative architecture is organized at two spatial scales: large domains, and smaller niches enriched for specific immune lineages. Some tumour cells express cell cycle regulators in the (canonical) patterns expected of freely growing cells, a phenomenon we refer to as 'cell cycle coherence'. By contrast, the cell cycles of other tumour cell populations are skewed towards specific phases or exhibit non-canonical (incoherent) marker combinations. Coherence varies across space, with changes in oncogene activity and therapeutic intervention, and is associated with aggressive tumour behaviour. Thus, multivariate measures from high-plex tissue images capture clinically significant features of cancer proliferation, a fundamental step in enabling more precise use of anti-cancer therapies.

STING agonism reprograms tumor-associated macrophages and overcomes resistance to PARP inhibition in BRCA1-deficient models of breast cancer.

PARP inhibitors (PARPi) have drastically changed the treatment landscape of advanced ovarian tumors with BRCA mutations. However, the impact of this class of inhibitors in patients with advanced BRCA-mutant breast cancer is relatively modest. Using a syngeneic genetically-engineered mouse model of breast tumor driven by Brca1 deficiency, we show that tumor-associated macrophages (TAMs) blunt PARPi efficacy both in vivo and in vitro. Mechanistically, BRCA1-deficient breast tumor cells induce pro-tumor polarization of TAMs, which in turn suppress PARPi-elicited DNA damage in tumor cells, leading to reduced production of dsDNA fragments and synthetic lethality, hence impairing STING-dependent anti-tumor immunity. STING agonists reprogram M2-like pro-tumor macrophages into an M1-like anti-tumor state in a macrophage STING-dependent manner. Systemic administration of a STING agonist breaches multiple layers of tumor cell-mediated suppression of immune cells, and synergizes with PARPi to suppress tumor growth. The therapeutic benefits of this combination require host STING and are mediated by a type I IFN response and CD8+ T cells, but do not rely on tumor cell-intrinsic STING. Our data illustrate the importance of targeting innate immune suppression to facilitate PARPi-mediated engagement of anti-tumor immunity in breast cancer.

How Compensatory Mechanisms and Adaptive Rewiring Have Shaped Our Understanding of Therapeutic Resistance in Cancer.

Therapeutic resistance to targeted therapies by tumor cells is a common and serious problem in the clinic. Increased understanding of the molecular mechanisms that underly resistance is necessary for the rational design and improvement of effective pharmacologic treatment strategies. The landmark study by O'Reilly and colleagues published in Cancer Research in 2006 provided valuable insights into nongenomic adaptive rewiring and compensatory mechanisms responsible for mediating resistance to targeted inhibition of the PI3K-AKT-mTOR pathway, and how tumor cells regulate signaling pathways via negative feedback loops. These findings have proven fundamental for guiding current efforts to develop effective combination treatments and provided a blueprint for research studies aimed at understanding the intricacies of cellular signaling.See related article by O'Reilly and colleagues, Cancer Res 2006;66:1500-8.

TAp63 is a transcriptional target of NF-kappaB.

The p53 homologue p63 encodes multiple protein isoforms either with (TA) or without (DeltaN) the N-terminal transactivation domain. Accumulating evidence indicates that TAp63 plays an important role in various biological processes, including cell proliferation, differentiation, and apoptosis. However, how TAp63 is regulated remains largely unclear. In this study, we demonstrate that NF-kappaB induces TAp63 gene expression. The responsible elements for NF-kappaB-mediated TAp63 induction are located within the region from -784 to -296 bp in the TAp63 promoter, which contains two NF-kappaB binding sites. Ectopic expression of RelA stimulates TAp63 promoter-driven reporter activity and increases endogenous TAp63 mRNA levels. Inhibition of NF-kappaB by IkappaBalpha super-repressor or with a chemical inhibitor leads to down regulation of TAp63 mRNA expression and activity. In addition, mutations in the critical NF-kappaB-binding sites significantly abolish the effects of NF-kappaB on TAp63. Activation of NF-kappaB by TNFalpha enhances p50/RelA binding to the NF-kappaB binding sites. Furthermore, we show that an Sp1 site adjacent to the NF-kappaB sites plays a role in NF-kappaB-mediated upregulation of TAp63. Taken together, these data reveal that TAp63 is a transcriptional target of NF-kappaB, which may play a role in cell proliferation, differentiation and survival upon NF-kappaB activation by various stimuli.

Integrating Immunotherapy and Targeted Therapy in Cancer Treatment: Mechanistic Insights and Clinical Implications.

Small-molecule targeted therapies have demonstrated outstanding potential in the clinic. These drugs are designed to minimize adverse effects by selectively attacking cancer cells while exerting minimal damage to normal cells. Although initial response to targeted therapies may be high, yielding positive response rates and often improving survival for an important percentage of patients, resistance often limits long-term effectiveness. On the other hand, immunotherapy has demonstrated durable results, yet for a limited number of patients. Growing evidence indicates that some targeted agents can modulate different components of the antitumor immune response. These include immune sensitization by inhibiting tumor cell-intrinsic immune evasion programs or enhancing antigenicity, as well as direct effects on immune effector and immunosuppressive cells. The combination of these two approaches, therefore, has the potential to result in synergistic and durable outcomes for patients. In this review, we focus on the latest advances on integrating immunotherapy with small-molecule targeted inhibitors. In particular, we discuss how specific oncogenic events differentially affect immune response, and the implications of these findings on the rational design of effective combinations of immunotherapy and targeted therapies.

Increased lysosomal biomass is responsible for the resistance of triple-negative breast cancers to CDK4/6 inhibition.

Inhibitors of cyclin-dependent kinases CDK4 and CDK6 have been approved for treatment of hormone receptor-positive breast cancers. In contrast, triple-negative breast cancers (TNBCs) are resistant to CDK4/6 inhibition. Here, we demonstrate that a subset of TNBC critically requires CDK4/6 for proliferation, and yet, these TNBC are resistant to CDK4/6 inhibition due to sequestration of CDK4/6 inhibitors into tumor cell lysosomes. This sequestration is caused by enhanced lysosomal biogenesis and increased lysosomal numbers in TNBC cells. We developed new CDK4/6 inhibitor compounds that evade the lysosomal sequestration and are efficacious against resistant TNBC. We also show that coadministration of lysosomotropic or lysosome-destabilizing compounds (an antibiotic azithromycin, an antidepressant siramesine, an antimalaria compound chloroquine) renders resistant tumor cells sensitive to currently used CDK4/6 inhibitors. Lastly, coinhibition of CDK2 arrested proliferation of CDK4/6 inhibitor-resistant cells. These observations may extend the use of CDK4/6 inhibitors to TNBCs that are refractory to current anti-CDK4/6 therapies.

Allele-Specific Chromatin Recruitment and Therapeutic Vulnerabilities of ESR1 Activating Mutations.

Estrogen receptor α (ER) ligand-binding domain (LBD) mutations are found in a substantial number of endocrine treatment-resistant metastatic ER-positive (ER+) breast cancers. We investigated the chromatin recruitment, transcriptional network, and genetic vulnerabilities in breast cancer models harboring the clinically relevant ER mutations. These mutants exhibit both ligand-independent functions that mimic estradiol-bound wild-type ER as well as allele-specific neomorphic properties that promote a pro-metastatic phenotype. Analysis of the genome-wide ER binding sites identified mutant ER unique recruitment mediating the allele-specific transcriptional program. Genetic screens identified genes that are essential for the ligand-independent growth driven by the mutants. These studies provide insights into the mechanism of endocrine therapy resistance engendered by ER mutations and potential therapeutic targets.

Metformin Sensitizes Leukemia Cells to Vincristine via Activation of AMP-activated Protein Kinase.

Vincristine is extensively used chemotherapeutic medicine to treat leukemia. However, it remains a critical clinical problem with regard to its toxicity and drug-resistance. AMP-activated protein kinase (AMPK) is an energy sensor that is pivotal in maintaining cell metabolic homeostasis. It is reported that AMPK is involved in vincristine-induced apoptosis. However, whether AMPK is involved in chemotherapy-resistance is largely unclear. It is well-documented that metformin, a widely used medicine to treat type II diabetes, possesses anti-cancer activities, yet whether metformin affects leukemia cell viability via vincristine is unknown. In this study, we showed that both AMPKα1 mRNA and phosphorylated AMPK protein levels were significantly decreased in clinical leukemia samples. We further demonstrated that metformin sensitized leukemia cells to vincristine-induced apoptosis in an AMPK-dependent manner. In addition, knockdown of AMPKα1 significantly reduced the effects of metformin on vincristine-induced apoptosis. Taken together, these results indicate that AMPK activation is critical in metformin effects on vincristine-induced apoptosis and suggest a putative strategy of a combination therapy using metformin and vincristine in treatment of leukemia.

MDM2/MDMX: Master negative regulators for p53 and RB.

MDM2 (mouse double minute 2 homolog) and MDMX (double minute X human homolog, also known as MDM4) are critical negative regulators of tumor protein p53. Our recent work shows that MDMX binds to and promotes degradation of retinoblastoma protein (RB) in an MDM2-dependent manner. In a xenograft tumor growth mouse model, silencing of MDMX results in inhibition of p53-deficient tumor growth, which can be effectively reversed by concomitant RB silencing. Thus, MDMX exerts its oncogenic activity via suppression of RB.

p53 and p73 Regulate Apoptosis but Not Cell-Cycle Progression in Mouse Embryonic Stem Cells upon DNA Damage and Differentiation.

Embryonic stem cells (ESCs) are fast proliferating cells capable of differentiating into all somatic cell types. In somatic cells, it is well documented that p53 is rapidly activated upon DNA damage to arrest the cell cycle and induce apoptosis. In mouse ESCs, p53 can also be functionally activated, but the precise biological consequences are not well characterized. Here, we demonstrated that doxorubicin treatment initially led to cell-cycle arrest at G2/M in ESCs, followed by the occurrence of massive apoptosis. Neither p53 nor its target gene p73 was required for G2/M arrest. Instead, p53 and p73 were fully responsible for apoptosis. p53 and p73 were also required for differentiation-induced apoptosis in mouse ESCs. In addition, doxorubicin treatment induced the expression of retinoblastoma protein in a p53-dependent manner. Therefore, both p53 and p73 are critical in apoptosis induced by DNA damage and differentiation.

p63α modulates c-Myc activity via direct interaction and regulation of MM1 protein stability.

Both p53-related p63 and c-Myc are transcription factors playing key roles in cell proliferation, survival, development and tumorigenesis. In the present study, we identified that MM1, a c-Myc inhibitor, specifically binds to C-termini of p63α (including ΔNp63α and TAp63α). Further study demonstrates that p63α facilitates MM1 protein degradation via proteasomal pathway, resulting in elevation of c-Myc transactivation activity. Knockdown of ΔNp63α leads to decrease in c-Myc protein levels, concomitant with reduced expression of CDK4 and Cyclin D1, and impaired cell cycle progression, both of which are effectively reversed by simultaneous knockdown of MM1. Moreover, expression of p63 and CDK4 is concomitantly up-regulated in B-cell acute lymphoblastic leukemia. Together, this study reveals a novel crosstalk between p63 and c-Myc that may play an important role in cell cycle progression and tumorigenesis.

Inhibition of Cdc42 is essential for Mig-6 suppression of cell migration induced by EGF.

The adaptor protein Mig-6 is a negative regulator of EGF signaling. It is shown that Mig-6 inhibits cell migration via direct interaction with the ErbB receptors, thereby inhibiting cross-phosphorylation or targeting the receptors for degradation. Mig-6 has also been shown to bind to and inhibit the Rho GTPase Cdc42 to suppress cytoskeletal rearrangement. However, the molecular mechanism(s) by which Mig-6 inhibits cell migration via Cdc42 is still not entirely clear. Here, we show that Mig-6 binding to Cdc42 is necessary and sufficient to inhibit EGF-induced filopodia formation and migration. This binding, mediated by four specific residues (I11, R12, M26, R30) in the Mig-6 CRIB domain, is essential for Mig-6 function. In addition, ectopic expression of Cdc42 reverses Mig-6 inhibition of cell migration. Mig-6 CRIB domain, alone, is sufficient to inhibit cell migration. Conversely, Mig-6 binding to EGFR is dispensable for Mig-6-mediated inhibition of cell migration. Moreover, we found that decreased Mig-6 expression correlates with cancer progression in breast and prostate cancers. Together, our results demonstrate that Mig-6 inhibition of Cdc42 signaling is critical in Mig-6 function to suppress cell migration and that dysregulation of this pathway may play a critical role in cancer development.