Although mutation of
has been described in non-small cell lung cancer (NSCLC), co-mutation with
, another Ras-GTPase activating protein (RasGAP), defines a novel genetically defined subclass of ...NSCLC.
/
-mutant cell lines are highly sensitive to MEK inhibitors, warranting clinical evaluation of MAPK inhibition in this subclass of patients.
.
An increasingly recognized component of resistance to tyrosine kinase inhibitors (TKI) involves persistence of a drug-tolerant subpopulation of cancer cells that survive despite effective eradication ...of the majority of the cell population. Multiple groups have demonstrated that these drug-tolerant persister cells undergo transcriptional adaptation via an epigenetic state change that promotes cell survival. Because this mode of TKI drug tolerance appears to involve transcriptional addiction to specific genes and pathways, we hypothesized that systematic functional screening of EGFR TKI/transcriptional inhibitor combination therapy would yield important mechanistic insights and alternative drug escape pathways. We therefore performed a genome-wide CRISPR/Cas9 enhancer/suppressor screen in EGFR-dependent lung cancer PC9 cells treated with erlotinib + THZ1 (CDK7/12 inhibitor) combination therapy, a combination previously shown to suppress drug-tolerant cells in this setting. As expected, suppression of multiple genes associated with transcriptional complexes (EP300, CREBBP, and MED1) enhanced erlotinib/THZ1 synergy. Unexpectedly, we uncovered nearly every component of the recently described ufmylation pathway in the synergy suppressor group. Loss of ufmylation did not affect canonical downstream EGFR signaling. Instead, absence of this pathway triggered a protective unfolded protein response associated with STING upregulation, promoting protumorigenic inflammatory signaling but also unique dependence on Bcl-xL. These data reveal that dysregulation of ufmylation and ER stress comprise a previously unrecognized TKI drug tolerance pathway that engages survival signaling, with potentially important therapeutic implications.
These findings reveal a novel function of the recently described ufmylation pathway, an ER stress survival signaling in drug-tolerant persister cells, which has important biological and therapeutic implications.
.
The Retinoblastoma (RB) tumor suppressor regulates G1/S transition during cell cycle progression by modulating the activity of E2F transcription factors. The RB pathway plays a central role in the ...suppression of most cancers, and RB mutation was initially discovered by virtue of its role in tumor initiation. However, as cancer genome sequencing has evolved to profile more advanced and treatment‐resistant cancers, it has become increasingly clear that, in the majority of cancers, somatic RB inactivation occurs during tumor progression. Furthermore, despite the presence of deregulation of cell cycle control due to an INK4A deletion, additional CCND amplification and/or other mutations in the RB pathway, mutation or deletion of the RB gene is often observed during cancer progression. Of note, RB inactivation during cancer progression not only facilitates G1/S transition but also enhances some characteristics of malignancy, including altered drug sensitivity and a return to the undifferentiated state. Recently, we reported that RB inactivation enhances pro‐inflammatory signaling through stimulation of the interleukin‐6/STAT3 pathway, which directly promotes various malignant features of cancer cells. In this review, we highlight the consequences of RB inactivation during cancer progression, and discuss the biological and pathological significance of the interaction between RB and pro‐inflammatory signaling.
Schematic presentation of the summary in this review. RB inactivation induces mitochondrial ROS production and subsequent activation of pro‐inflammatory and STAT3 loop in a JNK pathway dependent manner. Hypersecretion of IL‐6 is also post‐transcriptionally promoted by downregulation of mir‐140 following RB inactivation. Hypersecretion of pro‐inflammatory cytokines might contribute to establishment of inflammatory tumor microenvironment.
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Tumor Milieu Controlled by RB Tumor Suppressor Kitajima, Shunsuke; Li, Fengkai; Takahashi, Chiaki
International journal of molecular sciences,
2020-Apr-01, 2020-04-01, 20200401, Volume:
21, Issue:
7
Journal Article
Peer reviewed
Open access
The
gene is one of the most frequently mutated genes in human cancers. Canonically, RB exerts its tumor suppressive activity through the regulation of the G1/S transition during cell cycle ...progression by modulating the activity of E2F transcription factors. However, aberration of the
gene is most commonly detected in tumors when they gain more aggressive phenotypes, including metastatic activity or drug resistance, rather than accelerated proliferation. This implicates RB controls' malignant progression to a considerable extent in a cell cycle-independent manner. In this review, we highlight the multifaceted functions of the RB protein in controlling tumor lineage plasticity, metabolism, and the tumor microenvironment (TME), with a focus on the mechanism whereby RB controls the TME. In brief, RB inactivation in several types of cancer cells enhances production of pro-inflammatory cytokines, including CCL2, through upregulation of mitochondrial reactive oxygen species (ROS) production. These factors not only accelerate the growth of cancer cells in a cell-autonomous manner, but also stimulate non-malignant cells in the TME to generate a pro-tumorigenic niche in a non-cell-autonomous manner. Here, we discuss the biological and pathological significance of the non-cell-autonomous functions of RB and attempt to predict their potential clinical relevance to cancer immunotherapy.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Microfluidic culture has the potential to revolutionize cancer diagnosis and therapy. Indeed, several microdevices are being developed specifically for clinical use to test novel cancer therapeutics. ...To be effective, these platforms need to replicate the continuous interactions that exist between tumor cells and non-tumor cell elements of the tumor microenvironment through direct cell-cell or cell-matrix contact or by the secretion of signaling factors such as cytokines, chemokines and growth factors. Given the challenges of personalized or precision cancer therapy, especially with the advent of novel immunotherapies, a critical need exists for more sophisticated
ex vivo
diagnostic systems that recapitulate patient-specific tumor biology with the potential to predict response to immune-based therapies in real-time. Here, we present details of a method to screen for the response of patient tumors to immune checkpoint blockade therapy, first reported in Jenkins
et al. Cancer Discovery
, 2018,
8
, 196-215, with updated evaluation of murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS), including evaluation of the requirement for 3D microfluidic culture in MDOTS, demonstration of immune-checkpoint sensitivity of PDOTS, and expanded evaluation of tumor-immune interactions using RNA-sequencing to infer changes in the tumor-immune microenvironment. We also examine some potential improvements to current systems and discuss the challenges in translating such diagnostic assays to the clinic.
Microfluidic culture has the potential to revolutionize cancer diagnosis and therapy.
Cancer cell-intrinsic properties caused by oncogenic mutations have been well characterized; however, how specific oncogenes and tumor suppressors impact the tumor microenvironment (TME) is not well ...understood. Here, we present a novel non-cell-autonomous function of the retinoblastoma (RB) tumor suppressor in controlling the TME. RB inactivation stimulated tumor growth and neoangiogenesis in a syngeneic and orthotropic murine soft-tissue sarcoma model, which was associated with recruitment of tumor-associated macrophages (TAM) and immunosuppressive cells such as Gr1
CD11b
myeloid-derived suppressor cells (MDSC) or Foxp3
regulatory T cells (Treg). Gene expression profiling and analysis of genetically engineered mouse models revealed that RB inactivation increased secretion of the chemoattractant CCL2. Furthermore, activation of the CCL2-CCR2 axis in the TME promoted tumor angiogenesis and recruitment of TAMs and MDSCs into the TME in several tumor types including sarcoma and breast cancer. Loss of RB increased fatty acid oxidation (FAO) by activating AMP-activated protein kinase that led to inactivation of acetyl-CoA carboxylase, which suppresses FAO. This promoted mitochondrial superoxide production and JNK activation, which enhanced CCL2 expression. These findings indicate that the CCL2-CCR2 axis could be an effective therapeutic target in RB-deficient tumors. SIGNIFICANCE: These findings demonstrate the cell-nonautonomous role of the tumor suppressor retinoblastoma in the tumor microenvironment, linking retinoblastoma loss to immunosuppression.
Despite extensive efforts, oncogenic KRAS remains resistant to targeted therapy. Combined downstream RAL-TBK1 and MEK inhibition induces only transient lung tumor shrinkage in KRAS-driven genetically ...engineered mouse models (GEMMs). Using the sensitive KRAS;LKB1 (KL) mutant background, we identify YAP1 upregulation and a therapy-induced secretome as mediators of acquired resistance. This program is reversible, associated with H3K27 promoter acetylation, and suppressed by BET inhibition, resensitizing resistant KL cells to TBK1/MEK inhibition. Constitutive YAP1 signaling promotes intrinsic resistance in KRAS;TP53 (KP) mutant lung cancer. Intermittent treatment with the BET inhibitor JQ1 thus overcomes resistance to combined pathway inhibition in KL and KP GEMMs. Using potent and selective TBK1 and BET inhibitors we further develop an effective therapeutic strategy with potential translatability to the clinic.
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•LKB1 regulates innate immune signaling in KRAS-driven NSCLC cells•IGF1 and YAP1 promote resistance to TBK1/JAK/MEKi therapy in KL cells•BETi suppresses IGF1, YAP1 signaling and a therapy-induced secretome•Combination therapy with intermittent BETi achieves durable efficacy in KL models
Kitajima et al. identify BET-regulated YAP1 upregulation as a mediator of acquired and intrinsic resistance in KRAS;LKB1 and KRAS;TP53 mutant lung cancer cells, respectively, to combined TBK1 and MEK inhibition and show that intermittent BET inhibition overcomes this resistance.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Immunotherapy has shown limited efficacy in patients with EGFR-mutated lung cancer. Efforts to enhance the immunogenicity of EGFR-mutated lung cancer have been unsuccessful to date. Here, we discover ...that MET amplification, the most common mechanism of resistance to third-generation EGFR tyrosine kinase inhibitors (TKI), activates tumor cell STING, an emerging determinant of cancer immunogenicity (1). However, STING activation was restrained by ectonucleosidase CD73, which is induced in MET-amplified, EGFR-TKI-resistant cells. Systematic genomic analyses and cell line studies confirmed upregulation of CD73 in MET-amplified and MET-activated lung cancer contexts, which depends on coinduction of FOSL1. Pemetrexed (PEM), which is commonly used following EGFR-TKI treatment failure, was identified as an effective potentiator of STING-dependent TBK1-IRF3-STAT1 signaling in MET-amplified, EGFR-TKI-resistant cells. However, PEM treatment also induced adenosine production, which inhibited T-cell responsiveness. In an allogenic humanized mouse model, CD73 deletion enhanced immunogenicity of MET-amplified, EGFR-TKI-resistant cells, and PEM treatment promoted robust responses regardless of CD73 status. Using a physiologic antigen recognition model, inactivation of CD73 significantly increased antigen-specific CD8+ T-cell immunogenicity following PEM treatment. These data reveal that combined PEM and CD73 inhibition can co-opt tumor cell STING induction in TKI-resistant EGFR-mutated lung cancers and promote immunogenicity.
MET amplification upregulates CD73 to suppress tumor cell STING induction and T-cell responsiveness in TKI-resistant, EGFR-mutated lung cancer, identifying a strategy to enhance immunogenicity and improve treatment.
Studying metabolic directions and flow rates in cultured mammalian cells can provide key information for understanding metabolic function in the fields of cancer research, drug discovery, stem cell ...biology, and antibody production. In this work, metabolic engineering methodologies including medium component analysis, 13C-labeling experiments, and computer-aided simulation analysis were applied to characterize the metabolic phenotype of soft tissue sarcoma cells derived from p53-null mice. Cells were cultured in medium containing 1-13C glutamine to assess the level of reductive glutamine metabolism via the reverse reaction of isocitrate dehydrogenase (IDH). The specific uptake and production rates of glucose, organic acids, and the 20 amino acids were determined by time-course analysis of cultured media. Gas chromatography-mass spectrometry analysis of the 13C-labeling of citrate, succinate, fumarate, malate, and aspartate confirmed an isotopically steady state of the cultured cells. After removing the effect of naturally occurring isotopes, the direction of the IDH reaction was determined by computer-aided analysis. The results validated that metabolic engineering methodologies are applicable to soft tissue sarcoma cells derived from p53-null mice, and also demonstrated that reductive glutamine metabolism is active in p53-null soft tissue sarcoma cells under normoxia.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
One‐quarter of a century ago, identification of the human retinoblastoma gene (RB) loci proved Knudson's ‘two‐hit theory’ that tumor suppressor genes exist. Since then, numerous works delineated ...crucial roles for the RB protein (pRB)‐E2F transcription factor complex in G1‐S phase transition. In addition, discovering the relationship between pRB and tissue‐specific transcription factors enabled a better understanding of how cell cycle exit and terminal differentiation are coupled. Recent works provoked many exciting twists in views on pRB functions during cancer initiation and progression beyond its previously well‐appreciated roles. Various mitogenic and cytostatic cellular signals appeared to modulate pRB functions and thus affect a wide variety of effector molecules. In addition, genetic studies in mice as well as other creatures incessantly force us to revise our views on pRB functions. This review will focus particularly on the roles of pRB in regulating intracellular signaling, cell metabolism, chromatin function, stem cells and cancer stem cells. (Cancer Sci 2012; 103: 1182–1188)
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