Enhanced MAPK Signaling Research Articles

Enhanced MAPK signaling induced by CSF3R mutants confers dependence to DUSP1 for leukemic transformation

AbstractElevated MAPK and the JAK-STAT signaling play pivotal roles in the pathogenesis of chronic neutrophilic leukemia and atypical chronic myeloid leukemia. Although inhibitors targeting these pathways effectively suppress the diseases, they fall short in providing enduring remission, largely attributed to the cytostatic nature of these drugs. Even combinations of these drugs are ineffective in achieving sustained remission. Enhanced MAPK signaling besides promoting proliferation and survival triggers a proapoptotic response. Consequently, malignancies reliant on elevated MAPK signaling use MAPK feedback regulators to intricately modulate the signaling output, prioritizing proliferation and survival while dampening the apoptotic stimuli. Herein, we demonstrate that enhanced MAPK signaling in granulocyte colony-stimulating factor 3 receptor (CSF3R)–driven leukemia upregulates the expression of dual specificity phosphatase 1 (DUSP1) to suppress the apoptotic stimuli crucial for leukemogenesis. Consequently, genetic deletion of Dusp1 in mice conferred synthetic lethality to CSF3R-induced leukemia. Mechanistically, DUSP1 depletion in leukemic context causes activation of JNK1/2 that results in induced expression of BIM and P53 while suppressing the expression of BCL2 that selectively triggers apoptotic response in leukemic cells. Pharmacological inhibition of DUSP1 by BCI (a DUSP1 inhibitor) alone lacked antileukemic activity due to ERK1/2 rebound caused by off-target inhibition of DUSP6. Consequently, a combination of BCI with a MEK inhibitor successfully cured CSF3R-induced leukemia in a preclinical mouse model. Our findings underscore the pivotal role of DUSP1 in leukemic transformation driven by enhanced MAPK signaling and advocate for the development of a selective DUSP1 inhibitor for curative treatment outcomes.

Abstract B084: Sotorasib/SHP2 inhibitors combo, KRASG12C(ON)I and RASMULTI(ON)I effectively target KRASG12C tumors developing secondary resistance to Sotorasib via KRASG12C amplification

Abstract Background: Amplification of KRASG12C mutant allele has been reported as one of the resistance mechanisms in tumors progressed to Sotorasib, the FDA approved KRASG12C(OFF) inhibitor. An identical mechanism was observed in one of our H358 xenograft tumors relapsed to Sotorasib. The cell line generated from relapsed tumor represents an appropriate in vitro model to study therapeutic opportunities in KRASG12C tumors developing secondary resistance to Sotorasib via KRASG12C amplification. Methods: We generated five Sotorasib resistant cell lines using relapsed tumors of Sotorasib-treated H358 xenografts (MR1-MR5) and two vehicle-treated tumors (MV1 and MV2). Whole exome sequencing (WES), Taqman copy number analysis and digital PCR to study mutant allelic fractions were performed to investigate acquired mechanisms of resistance. We have also generated a Dox-inducible system for H358 cells to overexpress KRASG12C protein and mimic KRAS amplification. Results: We observed loss of sensitivity to Sotorasib in models MR1-MR5, when compared to MV1 and MV2. Western blot analysis show EMT-associated signatures in all resistant lines except MR2. The WES of resistant lines shows higher allelic frequency of KRASG12C mutation in MR2, which is homozygous (through loss of heterozygosity). The dPCR analysis further confirmed higher mutant allelic frequency in MR2. Copy-number analysis revealed the presence of more than 10 copies of KRAS in MR2. MR2 model represents the best model to investigate therapeutic options in KRASG12C tumors developing secondary resistance to Sotorasib via KRASG12C amplification. Both RAS-RAF Proximity Ligation and Raf-RBD pull down assays confirm enhanced RAS signaling in MR2 cells. The combination of KRASG12C(OFF) and SHP2 inhibitors, or single agent KRASG12C(ON) or RASMULTI(ON) inhibitors suppressed enhanced MAPK signaling in MR2 cells and drove tumor suppressions in MR2 mice xenografts. Conclusions: The stronger inhibition of KRAS pathway is necessary to suppress enhanced MAPK signaling in KRASG12C mutated lung cancer that developed secondary resistance to Sotorasib treatment through KRASG12C amplification. These preclinical data suggest that KRASG12C(OFF)/SHP2 inhibitors combo, or single agent KRASG12C(ON)I or RASMULTI(ON)I represent potential therapeutic strategies for these relapsed tumors. Citation Format: Hitendra S Solanki, Denis Imbody, Bina Desai, Ryoji Kato, Paul A Stewart, Yaakov Stern, Anurima Majumder, Liznair Bridenstine, Bhaswati Sarca, Daria Miroshnychenko, Ida Aronchik, Andriy Marusyk, Eric B Haura. Sotorasib/SHP2 inhibitors combo, KRASG12C(ON)I and RASMULTI(ON)I effectively target KRASG12C tumors developing secondary resistance to Sotorasib via KRASG12C amplification [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B084.

Extensive CXCR4-MAPK Signaling Activation in the Novel B Cell Acute Lymphoblastic Subtype of CDX2/Ubtf

Background B-cell acute lymphoblastic leukemia (B-ALL) is a genetically heterogeneous group of acute leukemia with novel subtypes emerging. Since we first reported a novel subgroup B-ALL with half cases possessing CXCR4 truncated mutations and abnormal expression of CDX2 in all cases, the genomic features were further elucidated by different research groups that concurrent focal deletions lead to both upstream enhancer hijacking by CDX2 and UBTF::ATXN7L3 fusion transcript. But the molecular characterization and underlying mechanism beyond the novel subtype B-ALL remain ambiguous. Methods In order to comprehensively elucidate the molecular features, we adopt whole genome sequencing to reveal the genomic abnormalities, including snv, indel, copy number variant, and structural variation. Then, the signaling transduction and functional effect of differentially expressed genes were conducted by transcriptome analysis. Furthermore, we constructed a weighted gene co-expression network (WGCNA) and transcriptional regulons network to unravel the hub genes or transcriptional factors in the subtype. Results We retrospectively identify 2.4% (n=12) cases belonging to a novel subtype among 503 B-ALL cases with available transcriptome data by unsupervised clustering, and this subtype is characterized by CXCR4 truncated mutations or high expression of FLNA which could enhance CXCR4 signaling. Among these patients, only one male was affected showing a strong sex tendency towards females. Uniformly, all cases harbor both focal deletions encompassing between PAN3 and FLT3 located upstream of CDX2, and between UBTF and ATXN7L3, unraveled by whole genome sequencing (n=10). The focal deletions lead to overexpression of CDX2 by enhancer hijacking of upstream PAN3 and fusion transcript UBTF::ATXN7L3, respectively. In addition, 80% (n=8) cases also occurred with 1q+ which is regarded as a factor contributing to poor prognosis in leukemia and lymphoma. Transcriptomic analysis of the differentially expressed genes (DEGs) for this subtype confirmed the critical role of oncogenic MAPK signaling. Enhanced MAPK signaling was further supported by the function of the hub genes including KIAA1549 and KIAA1549L, which belong to oncogenic MAPK signaling, derived from WGCNA analysis. According to the functional enrichment analysis of DEGs, axon guidance was the only significant enriched KEGG pathway. The hub genes NTN3 from WGCNA analysis and regulon CDX2 both confirmed the functional involvement of axon guidance in this subtype. Furthermore, we prospectively identify 8 more cases by RT-PCR screening of the UBTF::ATXN7L3 transcript. Totally, twenty patients have been found in our cohort, and interestingly, there exist two cases among this subtype accumulated with multiple subclones characterized by different CXCR4 mutations, which strengthen the genotoxic stress over CXCR4 signaling. Discussion Although genomic characterization of this novel subtype has been revealed by our and others' research. The functional effect and underlying mechanism have never been elucidated. Here, we emphasize that enhanced CXCR4 signaling in this subtype may act as an upstream driver event, and it could be supplied as a therapeutic target.

Abstract IA09: Allelic specificity: The key to NRAS-mutant melanoma initiation?

Abstract The reason why different NRAS mutants are enriched in each tumor type is unclear. We made a suite of conditional, Nras knock-in models (LSL-Q61R, -K, -L, -H, -P, -Q; G12D and G13D, -R) to test whether melanocyte transformation is dependent upon functions specific to the NRAS mutants found in human melanoma (Q61R and K). Spontaneous melanomas were rare in mice expressing Nras mutants infrequently observed in human melanoma (G12D, G13D, G13R, Q61H, or Q61P). However, melanocyte-specific Nras Q61R or Q61K expression induced rapid melanoma onset with high penetrance. Cohorts of mice carrying one LSL-Nras Q61R and one LSL-Nras Q61K, -L, -H, -P, or -Q allele were used to evaluate potential interactions between Nras mutants. These combinations initiated tumors with kinetics intermediate to those of mice homozygous for either allele (Nras Q61R or 61X) except in the case of Nras Q61Q/Q61R mice, which rarely developed tumors. Genomic and in vitro analyses revealed that the melanoma-initiating potential of each NRAS mutant directly correlated with enhanced MAPK signaling. Together, these data establish that functions specific to the most common NRAS mutants in human melanoma are needed to initiate the disease. Citation Format: Christin E. Burd. Allelic specificity: The key to NRAS-mutant melanoma initiation? [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA09.

Abstract 3871: KRAS G12C mutant allele amplification drives resistance to sotorasib in vitro

Abstract Sotorasib is an approved KRASG12C-selective inhibitor for the treatment of KRAS p.G12C-mutant advanced and previously treated non-small cell lung cancers (NSCLC). Acquired resistance due to genomic alterations following sotorasib treatment has been observed in 28% of lung cancer patients (CodeBreaK100, ASCO 2022) and can include bypass signaling, inactivation of negative feedback loops, or compensatory mutations in KRASG12C including amplification of KRAS. Preclinical studies on the mechanisms of acquired resistance are critical for understanding these resistance patterns and can reveal new therapeutic or combination strategies in the clinic. For that reason, we developed an NCI-H358 lung cancer model of acquired resistance to KRASG12C inhibition in vitro. NCI-H358 cells were continuously cultured in the presence of elevated concentrations of sotorasib (IC90) until resistance to KRASG12C inhibitor was achieved. Expression analyses of the resistant cells by ddPCR and immunoblotting showed increases in both KRAS gene and protein levels, resulting in enhanced MAPK signaling. siRNA knockdown of KRAS G12C expression re-sensitized the resistant cells to an analog of sotorasib. Further characterization of this cell line by whole exome sequencing (WES) and fluorescence in situ hybridization (FISH) showed that resistance was not due to genetic co-alterations but amplification of the mutant allele specifically. In comparison to DMSO-treated and parental controls, the sotorasib-resistant NCI-H358 cells contained an approximately 50-fold increase in KRAS G12C mutant allele copy numbers. Preclinical data generated using a KRASG12C inhibitor-resistant lung cancer cell line is consistent with clinical observations of acquired KRAS amplification following KRASG12C inhibitor treatment as a mechanism of resistance. This model further provides an opportunity to study acquired KRAS amplification and investigate combination treatment options in vitro. Citation Format: Deanna Mohn, Anne Y. Saiki, Pragathi Achanta, Andres Plata Stapper, J. Russell Lipford, Rati Verma. KRAS G12C mutant allele amplification drives resistance to sotorasib in vitro. [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 3871.

RAS oncogene signal strength regulates matrisomal gene expression and tumorigenicity of mouse keratinocytes.

Environmental and molecular carcinogenesis are linked by the discovery that chemical carcinogen induced-mutations in the Hras or Kras genes drives tumor development in mouse skin. Importantly, enhanced expression or allele amplification of the mutant Ras gene contributes to selection of initiated cells, tumor persistence, and progression. To explore the consequences of Ras oncogene signal strength, primary keratinocytes were isolated and cultured from the LSL-HrasG12D and LSL-KrasG12D C57BL/6J mouse models and the mutant allele was activated by adeno-Cre recombinase. Keratinocytes expressing one (H) or two (HH) mutant alleles of HrasG12D, one KrasG12D allele (K), or one of each (HK) were studied. All combinations of activated Ras alleles stimulated proliferation and drove transformation marker expression, but only HH and HK formed tumors. HH, HK, and K sustained long-term keratinocyte growth in vitro, while H and WT could not. RNA-Seq yielded two distinct gene expression profiles; HH, HK, and K formed one cluster while H clustered with WT. Weak MAPK activation was seen in H keratinocytes but treatment with a BRAF inhibitor enhanced MAPK signaling and facilitated tumor formation. K keratinocytes became tumorigenic when they were isolated from mice where the LSL-KrasG12D allele was backcrossed from the C57BL/6 onto the FVB/N background. All tumorigenic keratinocytes but not the non-tumorigenic precursors shared a common remodeling of matrisomal gene expression that is associated with tumor formation. Thus, RAS oncogene signal strength determines cell-autonomous changes in initiated cells that are critical for their tumor-forming potential.

CD59-Regulated Ras Compartmentalization Orchestrates Antitumor T-cell Immunity.

T cell-mediated immunotherapy represents a promising strategy for cancer treatment; however, it has achieved satisfactory clinical responses in only a limited population. Thus, a broader view of the T-cell immune response is required. The Ras/MAPK pathway operates in many important signaling cascades and regulates multiple cellular activities, including T-cell development, proliferation, and function. Herein, we found that the typical membrane-bound complement regulatory protein CD59 is located intracellularly in T cells and that the intracellular form is increased in the T cells of patients with cancer. When intracellular CD59 is abundant, it facilitates Ras transport to the inner plasma membrane via direct interaction; in contrast, when CD59 is insufficient or deficient, Ras is arrested in the Golgi, thus enhancing Ras/MAPK signaling and T-cell activation, proliferation, and function. mCd59ab deficiency almost completely abolished tumor growth and metastasis in tumor-bearing mice, in which CD4+ and CD8+ T cells were significantly increased compared with their proportions in wild-type littermates, and their proportions were inversely correlated with tumor growth. Using bone marrow transplantation and CD4+ and CD8+ T-cell depletion assays, we further demonstrated the critical roles of these cells in the potent antitumor activity induced by mCd59ab deficiency. Reducing CD59 expression also enhanced MAPK signaling and T-cell activation in human T cells. Therefore, the subcellular compartmentalization of Ras regulated by intracellular CD59 provides spatial selectivity for T-cell activation and a potential T cell-mediated immunotherapeutic strategy.

RASA2 ablation in T cells boosts antigen sensitivity and long-term function

The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints1,2. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function3–10. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in the bone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.

Abstract 1102: Fyn and Src cooperate with ERBB family to drive resistance to crizotinib in ROS1+ and ALK+ NSCLC

Abstract ALK/ROS1 TKIs have significantly improved outcomes for patients with ALK/ROS1+ lung adenocarcinoma. However, drug resistance inevitably develops, leading to disease progression. Approximately 1/3 of patients resistant to ROS1 TKIs demonstrate on-target ROS1 kinase domain (KD) mutations (Dziadziuszko et al. ESMO 2019), but the mechanism of resistance in most patients remains unknown/poorly characterized. To model and characterize acquired resistance to crizotinib, we used ROS1+ and ALK+ primary, patient tumor-derived non-small cell lung cancer (NSCLC) cell lines to generate acquired resistance models to the ALK/ROS1 inhibitor crizotinib. We used several techniques to probe the mechanisms driving resistance: DNA and RNA sequencing (seq), fluorescence in-situ hybridization (FISH), cell proliferation assays, and western blotting. The crizotinib-resistant line initially driven by a TPM3-ROS1 fusion (CUTO28-CR) displayed exquisite sensitivity to Src family kinase (SFK) TKIs in proliferation assays, which was modestly improved by addition of ERBB TKI afatinib. Bulk RNA seq revealed upregulation of SFKs Fyn and Src transcripts (141- and 2.31-fold, respectively). Interphase FISH revealed modest EGFR gene amplification in CUTO28-CR cells vs parental (EGFR:CEP7 ratio 2.1 vs 1.0), and RNA seq revealed upregulation of EGFR and HER2 transcripts (1.79- and 2.45-fold, respectively). Western blot analysis confirmed upregulation of Fyn at the protein level. CUTO28-CR cells treated with a SFK TKI revealed dose-dependent deactivation of Akt signaling, and addition of afatinib caused inhibition of MAPK signaling. Stimulation with epidermal growth factor (EGF) enhanced MAPK signaling in CUTO28-CR cells more than in parental cells, consistent with EGFR/HER2 dependence. Because of a noted propensity for the EML4-ALK line (H3122) to upregulate ERBB kinase activity under sustained ALK TKI treatment, we maintained these cells in low dose (25nM) afatinib while driving crizotinib resistance to identify additional bypass signaling pathways. These H3122-CAR cells were partially resensitized to crizotinib with the addition of SFK TKIs, and the addition of afatinib with SFK TKIs further resensitized these resistant cells to crizotinib. Bulk RNA seq of H3122-CAR cells revealed upregulation of Fyn and Src (2.64- and 3.63-fold, respectively), and western blot analysis confirmed upregulation at the protein level. In conclusion, we demonstrated increased SFK (particularly Fyn and/or Src) expression and/or activation is a mechanism utilized to drive bypass signaling, in cooperation with ERBB family kinases, to circumvent treatment with ALK/ROS1 TKI crizotinib. To our knowledge, this is the first report of SFK-driven bypass signaling as a mechanism of acquired resistance to an ALK/ROS1 TKI in NSCLC. The upregulation of Src and/or Fyn in NSCLC biopsies may guide the exploration of combination SFK TKIs with ALK/ROS1 TKIs. Citation Format: Logan C. Tyler, Nan Chen, Anh T. Le, Hala Nijmeh, Liming Bao, Robert C. Doebele. Fyn and Src cooperate with ERBB family to drive resistance to crizotinib in ROS1+ and ALK+ NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1102.

Abstract P076: Novel mechanisms of acquired TKI resistance in ROS1+ NSCLC

Abstract ROS1 tyrosine kinase inhibitors (TKIs) entrectinib and crizotinib have significantly improved outcomes for patients with ROS1 gene fusion positive (ROS1+) lung adenocarcinoma. However, drug resistance inevitably develops, leading to disease progression. Approximately one-third of patients resistant to ROS1 TKIs demonstrate on-target ROS1 kinase domain (KD) mutations (Dziadziuszko et al. ESMO 2019), but the mechanism of resistance in the majority of patients remain unknown or poorly characterized. The purpose of this study was to model and characterize mechanisms of acquired ROS1 TKI resistance. To investigate resistance to ROS1 TKIs, we used patient-derived cell lines CUTO28 (TPM3-ROS1) and CUTO37 (CD74-ROS1) to generate drug-resistant derivatives to entrectinib (CUTO28-ER, CUTO37-ER) or crizotinib (CUTO28-CR,CUTO37-CR) in vitro. We utilized several techniques to probe the mechanisms driving resistance, including DNA and RNA sequencing, fluorescence in-situ hybridization (FISH), cell proliferation assays, and western blotting. DNA sequencing of the ROS1 KD in all resistant cell lines did not reveal any mutations. CUTO28-ER cells displayed sensitivity to MET-selective TKIs in cell proliferation assays. Consistent with MET dependency, MAPK and Akt pathways were inhibited by treatment with a MET-selective, but not a ROS1-selective, TKI. Western blot demonstrated MET overexpression and interphase FISH confirmed MET gene amplification (MET:CEP7 ratio 4.2) compared to parental cells (MET:CEP7 ratio 1.0). Notably, metaphase FISH revealed extrachromosomal DNA (ecDNA) amplification of MET. CUTO28-CR cells displayed sensitivity to Src family kinase (SFK) TKIs, and addition of pan-HER TKI afatinib resulted in synergistic inhibition of proliferation and downstream MAPK signaling. CUTO37-ER cells displayed sensitivity via proliferation and signaling to pan-HER inhibitor afatinib, and phosphorylation of EGFR Y845 consistent with Src-mediated activation of EGFR. Parental CUTO28 and CUTO37 cells were not sensitive to SFK or HER inhibition. Interphase FISH revealed modest EGFR gene amplification in CUTO28-CR cells—(EGFR:CEP7 ratio 2.1) compared to parental (EGFR:CEP7 ratio 1.0)—but not in CUTO37-ER or CUTO37-CR cells. HER2 gene amplification was not identified in any of the cell lines. Stimulation with epidermal growth factor (EGF) markedly enhanced MAPK signaling in CUTO28-CR, CUTO37-ER, and CUTO37-CR cells to a greater extent than in corresponding parental cells, consistent with EGFR or HER2 dependence. In conclusion, we demonstrated novel mechanisms of resistance to ROS1 TKIs entrectinib and crizotinib. These include ecDNA amplification of MET as an alternate oncogene driver and Src/EGFR axis as a driver of bypass signaling. Combination strategies with existing TKIs could be explored in patients with ROS1 TKI resistance. The discovery of ecDNA MET amplification is particularly intriguing; ecDNA is associated with more aggressive cancers and next generation sequencing analyses do not typically involve amplicon reconstruction to detect ecDNA. Citation Format: Logan C. Tyler, Anh T. Le, Hala Nijmeh, Liming Bao, Kristen Turner, Jason Christianson, Robert C. Doebele. Novel mechanisms of acquired TKI resistance in ROS1+ NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P076.

KRASQ61H Preferentially Signals through MAPK in a RAF Dimer-Dependent Manner in Non–Small Cell Lung Cancer

Assembly of RAS molecules into complexes at the cell membrane is critical for RAS signaling. We previously showed that oncogenic KRAS codon 61 mutations increase its affinity for RAF, raising the possibility that KRASQ61H, the most common KRAS mutation at codon 61, upregulates RAS signaling through mechanisms at the level of RAS assemblies. We show here that KRASQ61H exhibits preferential binding to RAF relative to PI3K in cells, leading to enhanced MAPK signaling in in vitro models and human NSCLC tumors. X-ray crystallography of KRASQ61H:GTP revealed that a hyperdynamic switch 2 allows for a more stable interaction with switch 1, suggesting that enhanced RAF activity arises from a combination of absent intrinsic GTP hydrolysis activity and increased affinity for RAF. Disruption of KRASQ61H assemblies by the RAS oligomer-disrupting D154Q mutation impaired RAF dimerization and altered MAPK signaling but had little effect on PI3K signaling. However, KRASQ61H oligomers but not KRASG12D oligomers were disrupted by RAF mutations that disrupt RAF-RAF interactions. KRASQ61H cells show enhanced sensitivity to RAF and MEK inhibitors individually, whereas combined treatment elicited synergistic growth inhibition. Furthermore, KRASQ61H tumors in mice exhibited high vulnerability to MEK inhibitor, consistent with cooperativity between KRASQ61H and RAF oligomerization and dependence on MAPK signaling. These findings support the notion that KRASQ61H and functionally similar mutations may serve as predictive biomarkers for targeted therapies against the MAPK pathway. SIGNIFICANCE: These findings show that oncogenic KRASQ61H forms a cooperative RAS-RAF ternary complex, which renders RAS-driven tumors vulnerable to MEKi and RAFi, thus establishing a framework for evaluating RAS biomarker-driven targeted therapies.

Comprehensive analysis of Cry1Ac protoxin activation mediated by midgut proteases in susceptible and resistant Plutella xylostella (L.)

Insecticidal Cry toxins produced by Bacillus thuringiensis (Bt) have been widely used to control agricultural pests in both foliage sprays and transgenic crops. Nevertheless, rapid evolution of insect resistance to Cry toxins requires elucidation of the molecular mechanisms involved in Cry resistance. Two proposed models have been described to explain the toxicity of Cry proteins, the classic model states that Cry protoxin is activated by midgut proteases resulting in activated toxin that binds to receptors and forms a pore in the midgut cells triggering larval death, and the newly proposed dual model of the mode of action of Bt Cry toxins states that protoxin and activated toxins may have different mechanisms of action since several resistant strains to activated Cry toxins are still susceptible to the same Cry-protoxin. Protoxin activation by midgut proteases is a key step in both models. Herein, we evaluated Cry1Ac protoxin activation in a susceptible Plutella xylostella (L.) strain (DBM1Ac-S) and in the near-isogenic strain (NIL-R) with high field-evolved Cry1Ac resistance. Previous work showed that Cry1Ac resistance in NIL-R correlates with reduced binding to midgut receptors due to enhanced MAPK signaling pathway and down regulation of ABCC2 receptor. However, reduced midgut trypsin levels and altered midgut protease gene transcription were also observed in the Cry1Ac-resistant field isolated strain that is parent of the NIL-R strain. Therefore, we analyzed the midgut protease activities in both DBM1Ac-S and NIL-R strains. Detection of enzymatic activities showed that caseinolytic protease, trypsin and chymotrypsin activities were not significantly different between the susceptible and resistant strains. Furthermore, treatment with different trypsin or chymotrypsin inhibitors, such as Nα-tosyl-l-lysine chloromethyl ketone (TLCK) or Np-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not affect the susceptibility to Cry1Ac protoxin of the DBM1Ac-S and NIL-R larvae. Bioassay results indicated that the NIL-R larvae showed similar resistant levels to both Cry1Ac protoxin and trypsin-activated toxin. Taken together, our results demonstrated that high-level field-evolved Cry1Ac resistance in the NIL-R strain is independent of Cry1Ac protoxin activation and the specific protoxin mechanism of action. This discovery will strengthen our comprehensive understanding of the complex mechanistic basis of Bt resistance in different insects.

Tyrosine Threonine Kinase Inhibition Eliminates Lung Cancers by Augmenting Apoptosis and Polyploidy

The spindle assembly checkpoint maintains genomic integrity. A key component is tyrosine threonine kinase (TTK, also known as Mps1). TTK antagonism is hypothesized to cause genomic instability and cell death. Interrogating The Cancer Genome Atlas revealed high TTK expression in lung adenocarcinomas and squamous cell cancers versus the normal lung (P < 0.001). This correlated with an unfavorable prognosis in examined lung adenocarcinoma cases (P = 0.007). TTK expression profiles in lung tumors were independently assessed by RNA in situ hybridization. CFI-402257 is a highly selective TTK inhibitor. Its potent antineoplastic effects are reported here against a panel of well-characterized murine and human lung cancer cell lines. Significant antitumorigenic activity followed independent treatments of athymic mice bearing human lung cancer xenografts (6.5 mg/kg, P < 0.05; 8.5 mg/kg, P < 0.01) and immunocompetent mice with syngeneic lung cancers (P < 0.001). CFI-402257 antineoplastic mechanisms were explored. CFI-402257 triggered aneuploidy and apoptotic death of lung cancer cells without changing centrosome number. Reverse phase protein arrays (RPPA) of vehicle versus CFI-402257-treated lung cancers were examined using more than 300 critical growth-regulatory proteins. RPPA bioinformatic analyses discovered CFI-402257 enhanced MAPK signaling, implicating MAPK antagonism in augmenting TTK inhibitory effects. This was independently confirmed using genetic and pharmacologic repression of MAPK that promoted CFI-402257 anticancer actions. TTK antagonism exerted marked antineoplastic effects against lung cancers and MAPK inhibition cooperated. Future work should determine whether CFI-402257 treatment alone or with a MAPK inhibitor is active in the lung cancer clinic.

IGF1R as druggable target mediating PI3K-δ inhibitor resistance in a murine model of chronic lymphocytic leukemia

AbstractTargeted therapy is revolutionizing the treatment of cancers, but resistance evolves against these therapies and derogates their success. The phosphatidylinositol 3-kinase delta (PI3K-δ) inhibitor idelalisib has been approved for treatment of chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma, but the mechanisms conferring resistance in a subset of patients are unknown. Here, we modeled resistance to PI3K-δ inhibitor in vivo using a serial tumor transfer and treatment scheme in mice. Whole-exome sequencing did not identify any recurrent mutation explaining resistance to PI3K-δ inhibitor. In the murine model, resistance to PI3K-δ inhibitor occurred as a result of a signaling switch mediated by consistent and functionally relevant activation of insulin-like growth factor 1 receptor (IGF1R), resulting in enhanced MAPK signaling in the resistant tumors. Overexpression of IGF1R in vitro demonstrated its prominent role in PI3K-δ inhibitor resistance. IGF1R upregulation in PI3K-δ inhibitor–resistant tumors was mediated by functional activation and enhanced nuclear localization of forkhead box protein O1 transcription factors and glycogen synthase kinase 3β. In human CLL, high IGF1R expression was associated with trisomy 12. CLL cells from an idelalisib-treated patient showed decreased sensitivity to idelalisib in vitro concomitant with enhanced MAPK signaling and strong upregulation of IGF1R upon idelalisib exposure. Thus, our results highlight that alternative signaling cascades play a predominant role in the resistance and survival of cancer cells under PI3K-δ inhibition. We also demonstrate that these pathway alterations can serve as therapeutic targets, because inhibition of IGF1R offered efficacious salvage treatment of PI3K-δ inhibitor–resistant tumors in vitro and in vivo.

MiR-35 buffers apoptosis thresholds in the C. elegans germline by antagonizing both MAPK and core apoptosis pathways

Apoptosis is a genetically programmed cell death process with profound roles in development and disease. MicroRNAs modulate the expression of many proteins and are often deregulated in human diseases, such as cancer. C. elegans germ cells undergo apoptosis in response to genotoxic stress by the combined activities of the core apoptosis and MAPK pathways, but how their signalling thresholds are buffered is an open question. Here we show mir-35–42 miRNA family play a dual role in antagonizing both NDK-1, a positive regulator of MAPK signalling, and the BH3-only pro-apoptotic protein EGL-1 to regulate the magnitude of DNA damage-induced apoptosis in the C. elegans germline. We show that while miR-35 represses EGL-1 by promoting transcript degradation, repression of NDK-1 may be through sequestration of the transcript to inhibit translation. Importantly, dramatic increase in NDK-1 expression was observed in cells about to die. In the absence of miR-35, increased NDK-1 activity enhanced MAPK signalling that lead to significant increases in germ cell death. Our findings demonstrate that NDK-1 acts upstream of (or in parallel to) EGL-1, and that miR-35 targets both egl-1 and ndk-1 to fine-tune cell killing in response to genotoxic stress.

Vemurafenib impairs the repair of ultraviolet radiation-induced DNA damage.

Targeted therapy with the BRAF inhibitors vemurafenib and dabrafenib is an effective treatment regimen in patients with advanced melanoma carrying the BRAF V600E mutation. A common side effect is an enhanced rate of nonmelanoma skin cancer (NMSC). BRAF inhibition leads to a paradoxical enhanced MAPK signalling in BRAF wild-type cells, which might in part be responsible for the enhanced NMSC burden. It is known that disturbances of DNA repair result in an increased rate of NMSC. In the present study, it was investigated whether BRAF inhibitors might interfere with the repair of ultraviolet radiation-induced DNA damage in vitro. Epidermal keratinocytes of 11 Caucasian donors were treated with vemurafenib or dabrafenib and, 24 h later, exposed to ultraviolet A. DNA damage and repair capacity were analysed using south-western slot blot detecting cyclobutane pyrimidine dimers. Using PCR and DNA sequencing, RAS mutations and human papilloma virus genes were investigated. RNA expression was determined using a Gene Expression Chip and qRT-PCR. In 36% of keratinocytes, vemurafenib hampers the repair of ultraviolet A-induced DNA damage. No changes in DNA repair were observed with dabrafenib, indicating a possible substance-specific effect of vemurafenib. In none of the keratinocytes, pre-existing RAS mutations or human papilloma virus-associated DNA sequences were detected. The expression of the interferon-related damage resistance signature is decreased upon vemurafenib treatment in 36% of donors. The enhanced rate of NMSC in patients treated with vemurafenib might be partly related to a vemurafenib-driven impaired capacity for DNA repair.

CXCR5 overexpression in HL-60 cells enhances chemotaxis toward CXCL13 without anticipated interaction partners or enhanced MAPK signaling.

CXCR5 is a serpentine receptor implicated in cell migration in lymphocytes and differentiation in leukocytes. It causes MAPK pathway activation and has known membrane partners for signaling. CXCR5 mRNA is reportedly expressed in neutrophils following isolation, but its role in this cellular context is unknown. CXCR5 is also expressed in HL-60 cells, a human acute myeloid leukemia line, following treatment with all-trans retinoic acid, which induces differentiation toward a neutrophil-like state. CXCR5 is necessary for this process; differentiation was crippled in CXCR5 knockout cells and enhanced in cells ectopically expressing it. Since CXCR5 has various membrane protein partners, we investigated whether CXCR5-driven all-trans retinoic acid-induced differentiation depends on its association with such partners. Pursuing this, we generated HL-60 cells overexpressing the protein. We found that CXCR5 drove migration toward its ligand, CXCL13, and probed for interactions with several candidates using flow cytometry-based Förster resonance energy transfer. Surprisingly, we did not detect interactions with any candidates, including three reported in other cellular contexts. Additionally, we observed no significant changes in all-trans retinoic acid-induced differentiation; this may be due to the stoichiometry of CXCR5 and partner receptors or CXCL13. The anticipated membrane partnerings were surprisingly apparently unnecessary for downstream CXCR5 signaling and all-trans retinoic acid-induced differentiation.

MAPK Reliance via Acquired CDK4/6 Inhibitor Resistance in Cancer.

Purpose: Loss of cell-cycle control is a hallmark of cancer, which can be targeted with agents, including cyclin-dependent kinase-4/6 (CDK4/6) kinase inhibitors that impinge upon the G1-S cell-cycle checkpoint via maintaining activity of the retinoblastoma tumor suppressor (RB). This class of drugs is under clinical investigation for various solid tumor types and has recently been FDA-approved for treatment of breast cancer. However, development of therapeutic resistance is not uncommon.Experimental Design: In this study, palbociclib (a CDK4/6 inhibitor) resistance was established in models of early stage, RB-positive cancer.Results: This study demonstrates that acquired palbociclib resistance renders cancer cells broadly resistant to CDK4/6 inhibitors. Acquired resistance was associated with aggressive in vitro and in vivo phenotypes, including proliferation, migration, and invasion. Integration of RNA sequencing analysis and phosphoproteomics profiling revealed rewiring of the kinome, with a strong enrichment for enhanced MAPK signaling across all resistance models, which resulted in aggressive in vitro and in vivo phenotypes and prometastatic signaling. However, CDK4/6 inhibitor-resistant models were sensitized to MEK inhibitors, revealing reliance on active MAPK signaling to promote tumor cell growth and invasion.Conclusions: In sum, these studies identify MAPK reliance in acquired CDK4/6 inhibitor resistance that promotes aggressive disease, while nominating MEK inhibition as putative novel therapeutic strategy to treat or prevent CDK4/6 inhibitor resistance in cancer. Clin Cancer Res; 24(17); 4201-14. ©2018 AACR.

Elevated Mitochondrial Reactive Oxygen Species and Cellular Redox Imbalance in Human NADPH-Oxidase-Deficient Phagocytes.

Chronic granulomatous disease (CGD) is caused by mutations in genes that encode the NADPH-oxidase and result in a failure of phagocytic cells to produce reactive oxygen species (ROS) via this enzyme system. Patients with CGD are highly susceptible to infections and often suffer from inflammatory disorders; the latter occurs in the absence of infection and correlates with the spontaneous production of inflammatory cytokines. This clinical feature suggests that NADPH-oxidase-derived ROS are not required for, or may even suppress, inflammatory processes. Experimental evidence, however, implies that ROS are in fact required for inflammatory cytokine production. By using a myeloid cell line devoid of a functional NADPH-oxidase and primary CGD cells, we analyzed intracellular oxidants, signs of oxidative stress, and inflammatory cytokine production. Herein, we demonstrate that phagocytes lacking a functional NADPH-oxidase, namely primary CGD phagocytes and a gp91phox-deficient cell line, display elevated levels of ROS derived from mitochondria. Accordingly, these cells, despite lacking the major source of cellular ROS, display clear signs of oxidative stress, including an induced expression of antioxidants and altered oxidation of cell surface thiols. These observed changes in redox state were not due to abnormalities in mitochondrial mass or membrane integrity. Finally, we demonstrate that increased mitochondrial ROS enhanced phosphorylation of ERK1/2, and induced production of IL8, findings that correlate with previous observations of increased MAPK activation and inflammatory cytokine production in CGD cells. Our data show that elevated baseline levels of mitochondria-derived oxidants lead to the counter-intuitive observation that CGD phagocytes are under oxidative stress and have enhanced MAPK signaling, which may contribute to the elevated basal production of inflammatory cytokines and the sterile inflammatory manifestations in CGD.

DUSP1 is involved in the progression of small cell carcinoma of the prostate

Small cell carcinoma of the prostate (SCCP) is a rare and the most aggressive variant of prostate cancer. There is no effective cure or treatment for SCCP. Therefore, there is an urgent need for new therapy to improve the prognosis of patients with SCCP. DUSP1 is a dual specific phosphatase with an increasingly recognized in tumor biology. Altered expression of DUSP1 induced changes in the expression of genes involved in various biological pathways, including cell-cell signaling and angiogenesis. To understand more about the role of DUSP1 in SCCP, we evaluated the biological function and associated regulatory mechanism of DUSP1. In this study, DUSP1 was significantly down-regulated in human SCCP compared with the non-carcinoma tissues (P < 0.05). Overexpression of DUSP1 was found to suppress MAPK signaling and cell proliferation in PC-3 cells. Additionally, silencing of DUSP1 enhanced MAPK signaling and PC-3 cell proliferation. Moreover, it was observed that DUSP1 blocked the phosphorylation of p38 MAPK induced by anisomycin. Taken together, this investigation suggests that DUSP1 is involved in the progression of SCCP and may provide a new therapeutic target for SCCP treatment.