Activation Of MAP Kinase Pathway Research Articles

Targeting USP14/UCHL5: A Breakthrough Approach to Overcoming Treatment-Resistant FLT3-ITD-Positive AML.

FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations in acute myeloid leukemia (AML) are associated with poor prognosis and therapy resistance. This study aimed to demonstrate that inhibiting the deubiquitinating enzymes ubiquitin-specific peptidase 14 (USP14) and ubiquitin C-terminal hydrolase L5 (UCHL5) (USP14/UCHL5) with b-AP15 or the organogold compound auranofin (AUR) induces apoptosis in the ITD-transformed human leukemia cell line MV4-11 and mononuclear leukocytes derived from patients with FLT3-ITD-positive AML. This study included patients diagnosed with AML at Tokyo Medical and Dental University Hospital between January 2018 and July 2024. Both treatments blocked downstream FLT3 pathway events, with the effects potentiated by USP14 knockdown. Both treatments inhibited FLT3 deubiquitination via K48 and disrupted translation initiation via 4EBP1, a downstream FLT3 target. FLT3 was downregulated in the leukemic cells, with the associated activation of stress-related MAP kinase pathways and increased NF-E2-related factor 2. Furthermore, the overexpression of B-cell lymphoma-extra-large and myeloid cell leukemia-1 prevented the cell death caused by b-AP15 and AUR. These results suggest that inhibiting USP14/UCHL5, which involves multiple regulatory mechanisms, is a promising target for novel therapies for treatment-resistant FLT3-ITD-positive AML.

ΔNp63 bookmarks and creates an accessible epigenetic environment for TGFβ-induced cancer cell stemness and invasiveness

Backgroundp63 is a transcription factor with intrinsic pioneer factor activity and pleiotropic functions. Transforming growth factor β (TGFβ) signaling via activation and cooperative action of canonical, SMAD, and non-canonical, MAP-kinase (MAPK) pathways, elicits both anti- and pro-tumorigenic properties, including cell stemness and invasiveness. TGFβ activates the ΔNp63 transcriptional program in cancer cells; however, the link between TGFβ and p63 in unmasking the epigenetic landscape during tumor progression allowing chromatin accessibility and gene transcription, is not yet reported.MethodsSmall molecule inhibitors, including protein kinase inhibitors and RNA-silencing, provided loss of function analyses. Sphere formation assays in cancer cells, chromatin immunoprecipitation and mRNA expression assays were utilized in order to gain mechanistic evidence. Mass spectrometry analysis coupled to co-immunoprecipitation assays revealed novel p63 interactors and their involvement in p63-dependent transcription.ResultsThe sphere-forming capacity of breast cancer cells was enhanced upon TGFβ stimulation and significantly decreased upon ΔNp63 depletion. Activation of TGFβ signaling via p38 MAPK signaling induced ΔNp63 phosphorylation at Ser 66/68 resulting in stabilized ΔNp63 protein with enhanced DNA binding properties. TGFβ stimulation altered the ratio of H3K27ac and H3K27me3 histone modification marks, pointing towards higher H3K27ac and increased p300 acetyltransferase recruitment to chromatin. By silencing the expression of ΔNp63, the TGFβ effect on chromatin remodeling was abrogated. Inhibition of H3K27me3, revealed the important role of TGFβ as the upstream signal for guiding ΔNp63 to the TGFβ/SMAD gene loci, as well as the indispensable role of ΔNp63 in recruiting histone modifying enzymes, such as p300, to these genomic regions, regulating chromatin accessibility and gene transcription. Mechanistically, TGFβ through SMAD activation induced dissociation of ΔNp63 from NURD or NCOR/SMRT histone deacetylation complexes, while promoted the assembly of ΔNp63-p300 complexes, affecting the levels of histone acetylation and the outcome of ΔNp63-dependent transcription.ConclusionsΔNp63, phosphorylated and recruited by TGFβ to the TGFβ/SMAD/ΔNp63 gene loci, promotes chromatin accessibility and transcription of target genes related to stemness and cell invasion.

ASAH1 facilitates TNBC by DUSP5 suppression-driven activation of MAP kinase pathway and represents a therapeutic vulnerability

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that is prone to metastasis and therapy resistance. Owing to its aggressive nature and limited availability of targeted therapies, TNBC is associated with higher mortality as compared to other forms of breast cancer. In order to develop new therapeutic options for TNBC, we characterized the factors involved in TNBC growth and progression. Here, we demonstrate that N-acylsphingosine amidohydrolase 1 (ASAH1) is overexpressed in TNBC cells and is regulated via p53 and PI3K-AKT signaling pathways. Genetic knockdown or pharmacological inhibition of ASAH1 suppresses TNBC growth and progression. Mechanistically, ASAH1 inhibition stimulates dual-specificity phosphatase 5 (DUSP5) expression, suppressing the mitogen-activated protein kinase (MAPK) pathway. Furthermore, pharmacological cotargeting of the ASAH1 and MAPK pathways inhibits TNBC growth. Collectively, we unmasked a novel role of ASAH1 in driving TNBC and identified dual targeting of the ASAH1 and MAPK pathways as a potential new therapeutic approach for TNBC treatment.

Molecular mechanisms of the effects of mulberry (Morus Rubra) extracts on UV-radiation and oxidative stress-induced skin cell damage

Solar ultraviolet (UV) radiation, while beneficial for the homeostasis of human skin at proper doses, causes skin cell damage under extreme conditions, with a result of photoaging and skin cancer. With the depletion of the ozone layer, the prevalence of UV-induced skin cancer including melanoma has increased dramatically worldwide. Unfortunately, that poses a public health challenge for almost all countries. Over the past two decades, we have been working extensively on understanding the cellular and molecular mechanisms through which UV radiation induces skin aging and skin cancer. We have observed that UV radiation activates cell surface receptors, more pronouncedly EGFR, leading to a cascade of activation of MAP kinase pathways, with a result of MMP activation and in turn matrix degradation, and apoptosis. We have also found that UV radiation induces the downregulation of water channel aquaporins and skin cell dehydration. This novel discovery together with other reports has resulted in the emergence of numerous skin care products. Moreover, UV radiation induces the generation of reactive oxygen species, which exacerbates skin cell damage. With this important line of discovery, more skin care products with added antioxidants have been developed. Mulberries ( Morus Rubra) have been known to be rich in antioxidants and applied to cosmetic products in Asian countries with reported effcacy although the cellular and molecular mechanisms of its actions remain elusive. The objective of this study was to investigate these mechanisms from various perspectives. We hypothesize that the mulberry extracts affect cell signaling pathways that lead to protection against UV radiation or oxidative stressed-induced cell damage. First, we extracted the antioxidants with methanol from two types of mulberries, red and white. Human skin keratinocytes (HaCaT cells) were treated with UV radiation and hydrogen peroxide (H2O2), with and without mulberry extracts. Confocal microscopy and Western blot analysis were employed to measure various cell signaling components. The results showed that mulberry extracts protect against UV radiation and H2O2 induced cell death, with red extracts showing better protection than white extracts. Mulberry extracts attenuate H2O2-induced mitochondrial activity as tested by MitoTracker™ Red CMXRos. Our previous studies have shown that UV radiation induces mTOR pathway activation evidenced by elevation of phosphorylated S6. Interestingly, in this study, Western blot analysis data indicated that mulberry extracts potentiate UV-induced S6 phosphorylation and H2O2-induced MEK activation. Furthermore, confocal data demonstrated that mulberry extract pretreatment inhibits IL1β induced NFκB p65 translocation from the cytoplasm to the nucleus. Taken all together, our data suggest that mulberry extracts containing abundant antioxidants affect MAPK kinase and mTOR pathways and modulate cell signaling pathways associated with skin cell survival and inflammation under UV radiation and oxidative stress. Funding from the Committee on Aid to Faculty Research (CAFR) from Providence College. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Genomic and Transcriptomic Landscape of RET Wild-Type Medullary Thyroid Cancer and Potential Use of Mitogen-Activated Protein Kinase-Targeted Therapy.

About 75% of medullary thyroid cancers (MTCs) are sporadic with 45% to 70% being driven by a RET mutation. Selpercatinib is an approved treatment for RET-mutated (mut RET ) MTC; however, treatments are needed for wild-type RET MTC (wt RET ). Genomic alterations and transcriptomic signatures of wt RET MTC may reveal new therapeutic insights. We did a retrospective analysis of MTC samples submitted for DNA/RNA sequencing and programmed cell death ligand 1 expression using immunohistochemistry at a Clinical Laboratory Improvement Amendments/College of American Pathologists-certified laboratory. Tumor microenvironment immune cell fractions were estimated using RNA deconvolution (quanTIseq). Transcriptomic signatures of inflammation and MAP kinase pathway activation scores were calculated. Mann-Whitney U, chi-square, and Fisher's exact tests were applied (p values adjusted for multiple comparisons). The 160-patient cohort included 108 mut RET and 52 wt RET MTC samples. wt RET tumors frequently harbored mitogen-activated protein kinase (MAPK) pathway mutations, including HRAS (42.31%), KRAS (15.7%), NF1 (6.7%), and BRAF (2%), whereas only 1 MAPK pathway mutation ( NF1 ) was identified among mut RET MTC. Recurrent mutations seen in wt RET MTC included MGA , VHL, APC , STK11 , and NFE2L2 . Increased transcriptional activation of the MAPK pathway was observed in patients with wt RET harboring mutations in MAPK genes. Although the frequency of programmed cell death ligand 1 expression was similar in wt RET and mut RET (10.2% vs 7%, p = 0.531), wt RET tumors were more often tumor mutational burden high (7.7% vs 0%, p = 0.011), and wt RET MTC exhibited higher expression of immune checkpoint genes. We identified molecular alterations and immune-related features that distinguish wt RET from mut RET MTC. Although RET mutation drives MTC in the absence of other alterations, we showed that wt RET MTC frequently harbors MAPK pathway mutations. These findings may indicate a potential basis for MAPK-targeted therapy, possibly in combination with immuno-oncology agents for selected patients with wt RET MTC.

The E3 Ubiquitin Protein Ligase LINCR Amplifies the TLR-Mediated Signals through Direct Degradation of MKP1.

Toll-like receptors (TLRs) induce innate immune responses through activation of intracellular signaling pathways, such as MAP kinase and NF-κB signaling pathways, and play an important role in host defense against bacterial or viral infections. Meanwhile, excessive activation of TLR signaling leads to a variety of inflammatory disorders, including autoimmune diseases. TLR signaling is therefore strictly controlled to balance optimal immune response and inflammation. However, its balancing mechanisms are not fully understood. In this study, we identified the E3 ubiquitin ligase LINCR/ NEURL3 as a critical regulator of TLR signaling. In LINCR-deficient cells, the sustained activation of JNK and p38 MAPKs induced by the agonists for TLR3, TLR4, and TLR5, was clearly attenuated. Consistent with these observations, TLR-induced production of a series of inflammatory cytokines was significantly attenuated, suggesting that LINCR positively regulates innate immune responses by promoting the activation of JNK and p38. Interestingly, our further mechanistic study identified MAPK phosphatase-1 (MKP1), a negative regulator of MAP kinases, as a ubiquitination target of LINCR. Thus, our results demonstrate that TLRs fine-tune the activation of MAP kinase pathways by balancing LINCR (the positive regulator) and MKP1 (the negative regulator), which may contribute to the induction of optimal immune responses.

Antiviral responses induced by Tdap-IPV vaccination are associated with persistent humoral immunity to Bordetella pertussis

Many countries continue to experience pertussis epidemics despite widespread vaccination. Waning protection after booster vaccination has highlighted the need for a better understanding of the immunological factors that promote durable protection. Here we apply systems vaccinology to investigate antibody responses in adolescents in the Netherlands (N = 14; NL) and the United Kingdom (N = 12; UK) receiving a tetanus-diphtheria-acellular pertussis-inactivated poliovirus (Tdap-IPV) vaccine. We report that early antiviral and interferon gene expression signatures in blood correlate to persistence of pertussis-specific antibody responses. Single-cell analyses of the innate response identified monocytes and myeloid dendritic cells (MoDC) as principal responders that upregulate antiviral gene expression and type-I interferon cytokine production. With public data, we show that Tdap vaccination stimulates significantly lower antiviral/type-I interferon responses than Tdap-IPV, suggesting that IPV may promote antiviral gene expression. Subsequent in vitro stimulation experiments demonstrate TLR-dependent, IPV-specific activation of the pro-inflammatory p38 MAP kinase pathway in MoDCs. Together, our data provide insights into the molecular host response to pertussis booster vaccination and demonstrate that IPV enhances innate immune activity associated with persistent, pertussis-specific antibody responses.

CRISPR/Cas9-edited ROS1 + non-small cell lung cancer cell lines highlight differential drug sensitivity in 2D vs 3D cultures while reflecting established resistance profiles

IntroductionThe study of resistance-causing mutations in oncogene-driven tumors is fundamental to guide clinical decisions. Several point mutations affecting the ROS1 kinase domain have been identified in the clinical setting, but their impact requires further exploration, particularly in improved pre-clinical models. Given the scarcity of solid pre-clinical models to approach rare cancer subtypes like ROS1 + NSCLC, CRISPR/Cas9 technology allows the introduction of mutations in patient-derived cell lines for which resistant variants are difficult to obtain due to the low prevalence of cases within the clinical setting.MethodsIn the SLC34A2-ROS1 rearranged NSCLC cell line HCC78, we knocked-in through CRISPR/Cas9 technology three ROS1 drug resistance-causing mutations: G2032R, L2026M and S1986Y. Such variants are located in different functional regions of the ROS1 kinase domain, thus conferring TKI resistance through distinct mechanisms. We then performed pharmacological assays in 2D and 3D to assess the cellular response of the mutant lines to crizotinib, entrectinib, lorlatinib, repotrectinib and ceritinib. In addition, immunoblotting assays were performed in 2D-treated cell lines to determine ROS1 phosphorylation and MAP kinase pathway activity. The area over the curve (AOC) defined by the normalized growth rate (NGR_fit) dose–response curves was the variable used to quantify the cellular response towards TKIs.ResultsSpheroids derived from ROS1G2032R cells were significantly more resistant to repotrectinib (AOC fold change = − 7.33), lorlatinib (AOC fold change = − 6.17), ceritinib (AOC fold change = − 2.8) and entrectinib (AOC fold change = − 2.02) than wild type cells. The same cells cultured as a monolayer reflected the inefficacy of crizotinib (AOC fold change = − 2.35), entrectinib (AOC fold change = − 2.44) and ceritinib (AOC fold change = − 2.12) in targeting the ROS1 G2032R mutation. ROS1L2026M cells showed also remarkable resistance both in monolayer and spheroid culture compared to wild type cells, particularly against repotrectinib (spheroid AOC fold change = − 2.19) and entrectinib (spheroid AOC fold change = − 1.98). ROS1S1986Y cells were resistant only towards crizotinib in 2D (AOC fold change = − 1.86). Overall, spheroids showed an increased TKI sensitivity compared to 2D cultures, where the impact of each mutation that confers TKI resistance could be clearly distinguished. Western blotting assays qualitatively reflected the patterns of response towards TKI observed in 2D culture through the levels of phosphorylated-ROS1. However, we observed a dose–response increase of phosphorylated-Erk1/2, suggesting the involvement of the MAPK pathway in the mediation of apoptosis in HCC78 cells.ConclusionIn this study we knock-in for the first time in a ROS1 + patient-derived cell line, three different known resistance-causing mutations using CRISPR/Cas9 in the endogenous translocated ROS1 alleles. Pharmacological assays performed in 2D and 3D cell culture revealed that spheroids are more sensitive to TKIs than cells cultured as a monolayer. This direct comparison between two culture systems could be done thanks to the implementation of normalized growth rates (NGR) to uniformly quantify drug response between 2D and 3D cell culture. Overall, this study presents the added value of using spheroids and positions lorlatinib and repotrectinib as the most effective TKIs against the studied ROS1 resistance point mutations.

Activation of MAP Kinase Pathway by Polyisoprenylated Cysteinyl Amide Inhibitors Causes Apoptosis and Disrupts Breast Cancer Cell Invasion.

Prognoses for TNBC remain poor due to its aggressive nature and the lack of therapies that target its "drivers". RASA1, a RAS-GAP or GTPase-activating protein whose activity inhibits RAS signaling, is downregulated in up to 77% of TNBC cases. As such, RAS proteins become hyperactive and similar in effect to mutant hyperactive RAS proteins with impaired GTPase activities. PCAIs are a novel class of agents designed to target and disrupt the activities of KRAS and other G-proteins that are hyperactive in various cancers. This study shows the anticancer mechanisms of the PCAIs in two breast cancer cell lines, MDA-MB-468 and MDA-MB-231. PCAIs (NSL-YHJ-2-27) treatment increased BRAF phosphorylation, whereas CRAF phosphorylation significantly decreased in both cell lines. Moreover, the PCAIs also stimulated the phosphorylation of MEK, ERK, and p90RSK by 116, 340, and 240% in MDA-MB-468 cells, respectively. However, in MDA-MB-231 cells, a significant increase of 105% was observed only in p90RSK phosphorylation. Opposing effects were observed for AKT phosphorylation, whereby an increase was detected in MDA-MB-468 cells and a decrease in MDA-MB-231 cells. The PCAIs also induced apoptosis, as observed in the increased pro-apoptotic protein BAK1, by 51%, after treatment. The proportion of live cells in PCAIs-treated spheroids decreased by 42 and 34% in MDA-MB-468 and MDA-MB-231 cells, respectively, which further explains the PCAIs-induced apoptosis. The movement of the cells through the Matrigel was also inhibited by 74% after PCAIs exposure, which could have been due to the depleted levels of F-actin and vinculin punctate, resulting in the shrinkage of the cells by 76%, thereby impeding cell movement. These results show promise for PCAIs as potential therapies for TNBC as they significantly inhibit the hallmark processes and pathways that promote cell proliferation, migration, and invasion, which result in poor prognoses for breast cancer patients.

Genomics and transcriptomics of pancreatic adenosquamous carcinoma.

691 Background: Pancreatic cancer (PC) is anticipated to become the 2nd leading cause of cancer-related deaths by 2030. Pancreatic adenosquamous carcinoma (PASC) is a rare subtype of PC with histology including squamous adenomatous features (≥30% squamous histology), while pancreatic squamous cell cancer (PSCC) consists purely of squamous cells. We evaluated genomic, transcriptomic and prognostic differences between PASC, PSCC and pancreatic ductal adenocarcinoma (PDAC). Methods: 8951 PC tumors (83 PASC, 22 PSCC & 8846 PDAC) tested at Caris Life Sciences with WTS (Illumina, Novaseq) and NextGen DNA sequencing (NextSeq, 592 genes and NovaSeq, WES) and IHC were analyzed. Immune cell fraction was calculated by QuantiSeq. Overall survival (OS) analysis (time of tissue collection to last contact) was obtained from insurance claims and calculated with KM method. Statistical significance was determined using Chi-square/Fisher-Exact and adjusted for multiple comparisons (q<0.05). Results: No age or sex differences were seen between PDAC (median 68yo) and PASC (median 68.5yo), however PSCC was more prevalent in younger pts (median 62.5yo, q<0.05). Mutation rates of CDKN1B (3% vs 0.2%), SF3B1 (8 vs 2%), PTEN (5 vs 0.8%), BCL9 (4 vs 0.4%), AXIN1 (4 vs 0.2%) and CASP8 (3 vs 0.2%) and amplifications of AKT2 (5 vs 2%), ZNF384 (1 VS 0.1%), HMGA2 (7 vs 0.7%) and ROS1 fusions (1 vs 0.04%) all trended higher in PASC than PDAC (p<0.05). Several YAP signaling and squamous differentiation genes were significantly differentially expressed among histologies (table). PD-L1 IHC was higher in PASC vs PDAC (37 vs 14%, q<0.05); with a trend towards higher MSI-H (6 vs 1%) & TMB-H (6 vs 2%) in PASC vs PDAC (p<0.05). Higher infiltration of CD4+ T cells into the TME was seen in PASC vs PDAC (non-zero %, 73% vs 23%, q<0.05). Several immune genes ( CD274, IDO1, LAG3, & CTLA4) had higher expression in PASC vs PDAC (fold change 1.3-3.1, q<0.05). The MAP kinase pathway activation score (MPAS) was lower in PASC vs PDAC (median score -0.48 vs 0.25), while the IFN gamma signature was higher in PASC vs PDAC (median -0.24 vs -0.33) [both q<0.05]. There was increased OS for PASC compared to PSCC (12.6 vs 4.7 mo, HR=0.43, CI=0.25-0.75, p=0.002); no significant difference in OS was observed between PASC and PDAC. Conclusions: This is the largest molecular profiling analysis of PASC, which is characterized by unique genomic alterations, and is associated with higher PD-L1 expression, immune related gene expression, CD4+ T cell infiltration and IFN gamma signature, and lower MAPK activation. PASC is associated with better OS compared to PSCC. These findings may provide subtype-specific therapeutic opportunities for PASC and PSCC pts.[Table: see text]

Porcine Placenta Peptide Inhibits UVB-Induced Skin Wrinkle Formation and Dehydration: Insights into MAPK Signaling Pathways from In Vitro and In Vivo Studies.

Excessive exposure to ultraviolet (UV) radiation from sunlight accelerates skin aging, leading to various clinical manifestations such as wrinkles, dryness, and loss of elasticity. This study investigated the protective effects of porcine placenta peptide (PPP) against UVB-induced skin photoaging. Female hairless SKH-1 mice were orally administered PPP for 12 weeks, followed by UVB irradiation. PPP significantly reduced wrinkle formation, improved skin moisture levels, and prevented collagen degradation. Mechanistically, PPP inhibited the expression of matrix metalloproteinases (MMPs) and upregulated collagen production. Moreover, PPP elevated hyaluronic acid levels, contributing to enhanced skin hydration. Additionally, PPP demonstrated antioxidant properties by increasing the expression of the antioxidant enzyme GPx-1, thereby reducing UVB-induced inflammation. Further molecular analysis revealed that PPP suppressed the activation of p38 MAP kinase and JNK signaling pathways, crucial mediators of UV-induced skin damage. These findings highlight the potential of porcine placental peptides as a natural and effective intervention against UVB-induced skin photoaging. The study provides valuable insights into the mechanisms underlying the protective effects of PPP, emphasizing its potential applications in skincare and anti-aging formulations.

Preclinical Studies Demonstrating Efficacy of Tasquinimod in Models of Advanced Myeloproliferative Neoplasm (MPN) in Blastic Phase

The alarmins, S100A8 (A8) and S100A9 (A9), are low molecular weight proteins belonging to the S100 protein family. Also called calprotectins, A8 and A9 can form homodimers or heterodimers and bind Ca++. A8 and A9 are secreted into the extracellular space and plasma, where they interact with TLR4 (Toll like receptor 4), RAGE (Receptor for Advanced Glycation End products) and CD33. Intracellularly, they act as Ca++ biosensors and activate NADPH oxidase, stimulating ROS production, which activates NLRP3 inflammasome. A8 and A9 also amplify the inflammatory response through TLR4, and induce inflammatory cytokines, including TNFα and IL-6, through activation of NFkB and MAP kinase pathways. A8 and A9 are highly expressed in AML, especially myelomonocytic and monocytic AML, and are associated with poor prognosis. Through scRNA-Seq analysis, A8/A9 heterocomplex was shown to be highly expressed in myeloid and stromal cells and marked progression of bone marrow fibrosis in PMF (Primary Myelofibrosis). In the murine JAK2-V617F-driven PMF model, Tasquinimod (TM, Active Biotech), an orally active, quinoline-3-carboxamide linomide analogue immune-modulatory agent was shown to bind A9, inhibiting its interaction with TLR and RAGE receptors. Notably, TM treatment reduced myeloproliferation, splenomegaly and MF in JAK2-V617F mice. Therefore, based on these findings, in present studies, we determined in vitro and in vivo efficacy of TM against cellular models of advanced MPN cell lines and patient-derived (PD) CD34+ blastic phase (BP, > 5% blasts in PB) MPN cells. Treatment with TM (10 to 30 µM for 96 hours) induced loss of viability in the MPN-AML SET2, HEL92.1.7 cell lines and PD MPN-BP cells, but not in normal CD34+ progenitor cells. RNA-Seq analysis showed that exposure to TM (20 µM for 16 hours) led to negative enrichment of the gene-sets of MYC and E2F targets, of mTORC1 and IL6-JAK-STAT3 signaling, and of inflammatory response in PD MPN-AML cells. This was associated with log2 fold-decline in MYC, BCL2, CDK6, MPL, CCND1 and myeloperoxidase (MPO), but upregulation of mRNA of GFI1 and p21. CyTOF analysis revealed that TM treatment significantly reduced expressions of A8, A9, p-ERK, MPO, CXCR4, Cyclin D1, PU.1 and Ki67, but increased protein levels of GFI1, p21 and cleaved PARP in phenotypically defined PD, CD34+ MPN-BP cells expressing high levels of CLEC12A, CD99, CD123 but low expression of CD11b. Additionally, our findings showed that co-treatment with TM (5 to 30 µM) and ruxolitinib (250 to 1000 nM), BET inhibitor OTX015 (50 to 250 nM) or pelabresib (CPI-0610) (100 to 500 nM), or BCL2/Bcl-xL inhibitor navitoclax induced synergistic lethality in advanced MPN-BP cells exhibiting delta synergy scores of >1.0 (by the ZIP method). Notably, our findings also demonstrated that treatment with TM (10 or 30 mg/kg/day, by oral gavage) for 8-weeks, significantly improved survival without inducing any toxicity in immune-depleted NSG mice engrafted with PDX cells of MPN-AML with mutations in CALR and TERT. In a separate experiment on the same PDX model, treatment with TM (30 mg/kg/day) also induced significantly greater survival advantage than treatment with ruxolitinib (30 mg/kg/day) or OTX015 (30 mg/kg/day) by oral gavage. These findings clearly demonstrate preclinical efficacy of TM in advanced MPN-BP cellular models and create the rationale to further interrogate the efficacy of TM alone and in combinations with current, front-line therapies for advanced MPN with excess blasts.

Abstract CT242: A phase I study of ruxolitinib in combination with abemaciclib for patients with primary or post-polycythemia vera/essential thrombocythemia myelofibrosis

Abstract Introduction: Patients (pts) with primary and secondary myelofibrosis (MF) have a reduced life expectancy. While several oral Janus Kinase (JAK)-1/2 inhibitors such as ruxolitinib (RUX) have demonstrated improvements in symptom burden and spleen volume reduction compared to physician choice of best available therapy leading to their approval by the US FDA, responses to these agents are generally time-limited. Additionally, these agents are not considered to be disease-modifying and survival in pts after RUX discontinuation is poor with a median survival of 11-14 months. Thus, novel mechanism-based therapies for pts with suboptimal response or progression on RUX are needed. Preclinical Rationale: JAK2 V617F mutations promote transition from G1 to S-phase of the cell cycle via increased expression of CDC25A in preclinical models and CDC25A expression is upregulated in primary MPN patient samples. Additional cell cycle regulators such as CDK6 and Cyclin D also play a role in MF pathogenesis and drive myeloproliferation. Finally, combination of the CDK4/6 inhibitor ribociclib and RUX demonstrated reduction in spleen and liver size when compared to RUX alone in murine models, suggesting synergy (Rampal et al., CCR 2021). Methods: This multicenter, phase I dose-escalation trial evaluates the safety of RUX + the CDK4/6 inhibitor abemaciclib in pts with primary or secondary MF with intermediate-1/2 or high-risk disease by DIPSS who require treatment and had an inadequate response to RUX. An inadequate response will be defined by (I) palpable splenomegaly ≥5 cm below the left costal margin at study entry AND/OR (II) active MPN symptoms [presence of 1 symptom score ≥5 or 2 symptom scores ≥3 using the screening MPN-SAF TSS. This study follows a conventional “3+3” dose-escalation design, in which pts on a stable dose of RUX (10mg or 15mg BID) with sufficient baseline platelet and absolute neutrophil count will be treated with increasing doses of abemaciclib. Abemaciclib was chosen due to less myelosuppression compared with other CDK4/6 inhibitors. The pre-study dose of ruxolitinib will be maintained to determine the maximum tolerated dose of the combination of RUX+ abemaciclib. The primary endpoint is to determine the safety and tolerability, and to identify the recommended phase II dose of abemaciclib in MF pts on a stable dose of RUX. Secondary endpoints include overall response rate, progression-free and overall survival, and duration of response per the modified International Working Group - Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) and the European Leukemia Net (ELN) 2013 consensus definitions. Correlative studies are planned to identify biomarkers predictive of response to RUX + abemaciclib, mechanisms of resistance (e.g., MAP kinase pathway activation), and any disease-modifying effects of combination therapy. Citation Format: Jan P. Bewersdorf, Srdan Verstovsek, Andriy Derkach, Lucia Masarova, Naveen Pemmaraju, Eytan M. Stein, Michael Mauro, Raajit Rampal, Prithviraj Bose. A phase I study of ruxolitinib in combination with abemaciclib for patients with primary or post-polycythemia vera/essential thrombocythemia myelofibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT242.

Myeloid cells from Langerhans cell histiocytosis patients exhibit increased vesicle trafficking and an altered secretome capable of activating NK cells.

Langerhans cell histiocytosis (LCH) is a potentially life-threatening inflammatory myeloid neoplasia linked to pediatric neurodegeneration, whereby transformed LCH cells form agglomerated lesions in various organs. Although MAP-kinase pathway mutations have been identified in LCH cells, the functional consequences of these mutations and the mechanisms that cause the pathogenic behavior of LCH cells are not well understood. In our study, we used an in vitro differentiation system and RNA-sequencing to compare monocyte-derived dendritic cells from LCH patients to those derived from healthy controls or patients with Crohn's disease, a non-histiocytic inflammatory disease. We observed that interferon-γ treatment exacerbated intrinsic differences between LCH patient and control cells, including strikingly increased endo- and exocytosis gene activity in LCH patients. We validated these transcriptional patterns in lesions and functionally confirmed that LCH cells exhibited increased endo- and exocytosis. Furthermore, RNA-sequencing of extracellular vesicles revealed the enrichment of pathological transcripts involved in cell adhesion, MAP-kinase pathway, vesicle trafficking and T-cell activation in LCH patients. Thus, we tested the effect of the LCH secretome on lymphocyte activity and found significant activation of NK cells. These findings implicate extracellular vesicles in the pathology of LCH for the first time, in line with their established roles in the formation of various other tumor niches. Thus, we describe novel traits of LCH patient cells and suggest a pathogenic mechanism of potential therapeutic and diagnostic importance.

Brain Natriuretic Peptide Protects Cardiomyocytes from Apoptosis and Stimulates Their Cell Cycle Re-Entry in Mouse Infarcted Hearts.

Brain Natriuretic Peptide (BNP) supplementation after infarction increases heart function and decreases heart remodeling. BNP receptors, NPR-A and NPR-B are expressed on adult cardiomyocytes (CMs). We investigated whether a part of the BNP cardioprotective effect in infarcted and unmanipulated hearts is due to modulation of the CM fate. For this purpose, infarcted adult male mice were intraperitoneally injected every two days during 2 weeks with BNP or saline. Mice were sacrificed 1 and 14 days after surgery. BNP or saline was also injected intraperitoneally every two days into neonatal pups (3 days after birth) for 10 days and in unmanipulated 8-week-old male mice for 2 weeks. At sacrifice, CMs were isolated, counted, measured, and characterized by qRT-PCR. The proportion of mononucleated CMs was determined. Immunostainings aimed to detect CM re-entry in the cell cycle were performed on the different hearts. Finally, the signaling pathway activated by BNP treatment was identified in in vitro BNP-treated adult CMs and in CMs isolated from BNP-treated hearts. An increased number of CMs was detected in the hypoxic area of infarcted hearts, and in unmanipulated neonatal and adult hearts after BNP treatment. Accordingly, Troponin T plasma concentration was significantly reduced 1 and 3 days after infarction in BNP-treated mice, demonstrating less CM death. Furthermore, higher number of small, dedifferentiated and mononucleated CMs were identified in adult BNP-treated hearts when compared to saline-treated hearts. BNP-treated CMs express higher levels of mRNAs coding for hif1 alpha and for the different cyclins than CMs isolated from saline-treated hearts. Higher percentages of CMs undergoing DNA synthesis, expressing Ki67, phospho histone3 and Aurora B were detected in all BNP-treated hearts, demonstrating that CMs re-enter into the cell cycle. BNP effect on adult CMs in vivo is mediated by NPR-A binding and activation of the ERK MAP kinase pathway. Interestingly, an increased number of CMs was also detected in adult infarcted hearts treated with LCZ696, an inhibitor of the natriuretic peptide degradation. Altogether, our results identified BNP and all therapies aimed to increase BNP's bioavailability as new cardioprotective targets as BNP treatment leads to an increased number of CMs in neonatal, adult unmanipulated and infarcted hearts.

Phase I Study of Ruxolitinib in Combination with Abemaciclib for Patients with Primary or Post-Polycythemia Vera/Essential Thrombocythemia Myelofibrosis

Introduction and preclinical rationale: Patients with primary and secondary myelofibrosis (MF) have a reduced life expectancy compared to age-matched controls. The prognosis of individual patients is defined by clinical, cytogenetic, and molecular disease characteristics. Several oral Janus Kinase (JAK)-1/2 inhibitors such as ruxolitinib are approved by the US FDA for the treatment of patients with intermediate or high-risk MF based on improvements in symptom burden and spleen volume reduction compared to physician choice of best available therapy. However, these inhibitors often have a limited duration of response, and survival in patients after ruxolitinib discontinuation is poor (median survival of 13 months). Novel mechanism-based therapies for patients with suboptimal response or progression on ruxolitinib are thus needed. Preclinical studies have demonstrated that JAK2 V617F mutations promote transition from G1 to S-phase of the cell cycle via increased expression of CDC25A. Notably, CDC25A expression has been shown to be upregulated in primary MPN patient samples. Additional cell cycle regulators such as CDK6 and Cyclin D have also been demonstrated to play a role in MPN pathogenesis and to drive myeloproliferation. Preclinical studies testing the combination of the CDK4/6 inhibitor ribociclib and ruxolitinib demonstrated reduction in spleen and liver size when compared to ruxolitinib alone in murine models, suggesting synergism (Rampal et al., Clinical Cancer Research 2021). Methods: Based on these promising preclinical data, we hypothesize that combining ruxolitinib with a CDK4/6 inhibitor could have synergistic effects in patients with primary or secondary MF who have an inadequate response to ruxolitinib monotherapy. In this multicenter, phase I dose-escalation trial we evaluate the safety of combination therapy with ruxolitinib and the CDK4/6 inhibitor abemaciclib. Patients with primary or secondary MF with intermediate-1/2 or high-risk disease by DIPSS who require treatment and had an inadequate response to ruxolitinib are eligible for enrollment. An inadequate response will be defined by (I) palpable splenomegaly ≥5 cm below the left costal margin at study entry AND/OR (II) active MPN symptoms [presence of one symptom score ≥5 or two symptom scores ≥3 using the screening MPN-SAF TSS (Emanuel at al. JCO 2012). (Table 1). This study follows a conventional "3+3” dose-escalation design, in which patients on a stable dose of ruxolitinib (10mg or 15mg BID) with sufficient baseline platelet and absolute neutrophil count will be treated with increasing doses of abemaciclib. The pre-study dose of ruxolitinib will be maintained to determine the maximum tolerated dose of the combination of ruxolitinib and abemaciclib (Figure 1). The primary endpoint of this study is to determine the safety and tolerability, and to identify the recommended phase II dose of abemaciclib in MF patients on a stable, standard dose of ruxolitinib. Secondary endpoints include overall response rate (defined as a composite of complete remission [CR], partial remission [PR], and clinical improvement [CI; defined as anemia, spleen or symptom response]), rates of CR, PR, and CI as well as progression-free survival (PFS), overall survival (OS), and duration of response (DOR) as defined in accordance with the modified 2013 consensus definitions proposed by the International Working Group - Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) and the European Leukemia Net (ELN). As part of this clinical trial several correlative studies are planned to identify biomarkers predictive of response to ruxolitinib and abemaciclib as well as mechanisms of resistance (e.g., MAP kinase pathway activation). These include evaluation of changes in variant allele fraction of driver mutations, gene expression profile, cell cycle, clonal architecture, cytokine profile, and bone marrow fibrosis, aiming to inform patient selection as well as other alternative combination therapies for future clinical trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

1771P Genomic characteristics and clinical outcomes of HRAS-mutated urothelial bladder cancer

HRAS gene encodes the GTPase HRAS protein, a member of the RAS superfamily, which is enriched in high-grade urothelial bladder carcinoma (UBC). We aimed to characterize genomic and clinical outcomes of patients with HRAS mutations (HRASmt) as a possible prognostic and therapeutic biomarker. A total of 3,426 UBC tissue samples underwent molecular profiling at Caris Life Sciences utilizing NGS of DNA. MAP kinase pathway activation and the likelihood of a tumor’s response to anti-PD(L)1 therapy were measured via MPAS and IFN signature, respectively. Wilcoxon, Fisher’s exact were used for statistical significance. Overall survival (OS) was calculated from the date of tissue collection or the start of treatment until the last contact from insurance claims. All comparisons were conducted between HRAS mutated tumors and the entire UBC general cohort (GC). HRASmt were detected in 107 patients with metastatic UBC (3.12%). Of those, 70% were detected in primary and 30% from metastatic sites. HRASmt were associated with lower TMB (5 vs 8 mut/Mb, q<0.01) and higher PD-L1 expression (55.3% vs 39.1%, q<0.01). HRASmt tumors harbored less TP53 (25.5% vs 60.1%, q<0.05), RB1 (5.3% vs 22.1%, q<0.05), FGFR3 (1.9% vs 13.9%, q<0.05), and KMT2D (14.0% vs 25.4%, q<0.05), but more BRAF mutations (14.0% vs 2.1%, q<0.05). The most frequent HRAS point mutation involved Q61 (47.6%). HRAS-Q61 mutant tumors showed higher MPAS scores (2.21 vs 0.6, q<0.001), enrichment in pathways (all q<0.05) such as EMT (0.86 vs 0.83), TGFb (0.91 vs 0.89), angiogenesis (0.78 vs 0.75) and notch signaling (0.82 vs 0.81). Patients with HRAS-Q61 point mutations experienced worse OS and prognosis compared to HRASwt tumors when treated with checkpoint inhibitors (HR 1.45, 95% 1.01-2.10; p <0.05). HRAS-Q61 point mutations represent a clinically relevant mutation in UBC, as evidenced by its association with worse clinical outcomes compared to HRASwt tumors. Our transcriptomic analysis suggests HRAS-Q61 mutations were associated with lower response to immunotherapy and leads to MAPK activation, as well as modulation of important TME pathways, possibly indicating a distinct biological and clinical phenotype. Our study provides rationale for therapeutic HRAS targeting in UBC.

Melatonin mediated activation of MAP kinase pathway may reduce DNA damage stress in plants: A review.

Melatonin is an important biomolecule found in diverse groups of organisms. Under different abiotic stresses, the synthesis of melatonin is markedly increased suggesting pivotal roles of melatonin in plants enduring stresses. Being an endogenous signaling molecule with antioxidant activity, melatonin alters many physiological responses and is found to be involved in regulating DNA damage responses. However, the molecular mechanisms of melatonin in response to DNA damage have not yet been studied. The present review aims to provide insights into the molecular mechanisms of melatonin in response to DNA damage in plants. We propose that the MAP kinase pathway is involved in regulating melatonin dependent response of plants under DNA damage stress. Where melatonin might activate MAPK via H2 O2 or Ca2+ dependent pathways. The activated MAPK in turn might phosphorylate and activate SOG1 and repressor type MYBs to mitigate DNA damage under abiotic stress.

Abstract 2683: Activation of MAP kinase pathway in breast cancer cell lines by polyisoprenylated cysteinyl amide inhibitors (PCAIs) causes cell death

Abstract Female breast cancer has now surpassed lung cancer as the most common cancer overall in both sexes with an estimated 2.3 million new cases, representing 11.7% of all cancer cases worldwide in 2020. Triple negative breast cancer (TNBC) has been the subject of intensive studies due to its aggressive nature which makes it difficult to treat. Since inclusive treatment for this type had been elusive for decades, it is thus imperative to develop new therapeutic agents to mitigate its deadly effects. PCAIs are novel compounds designed to mimic the essential posttranslational modifications of G-proteins such as KRAS. This study elucidates the effect of the PCAIs downstream of KRAS, specifically on the mitogen activated pathway kinase (MAPK) enzymes. Two breast cancer cell lines, MDA-MB-468 and MDA-MB-231, were used to determine the levels of phosphorylation in several MAPK pathway enzymes. After exposure of the cells to varying concentrations of PCAIs (NSL-YHJ-2-27) for 48 hours, western blot results showed remarkable differences in the phosphorylated levels of the respective enzymes in cells treated with 5 µm of the PCAIs as compared to the control (NSL-YHJ-2-62). Interestingly, the cell lines reacted differently after treatment, in which MDA-MB-468 cells showed increased levels of phosphorylated MEK, ERK, and 90RSK by 122, 273 and 242%, respectively. However, in MDA-MB-231, only the phosphorylated 90RSK showed an increase of 105% and no changes in the other two enzymes. Moreover, a very distinct difference was observed in the levels of phosphorylated BRAF (p-BRAF) and CRAF (p-CRAF). In MDA-MB-231 cells, no change in p-BRAF levels while a 184% increase in p-CRAF were observed. In MDA-MB-468, p-BRAF increased by 41% and p-CRAF decreased by 58%. The differences in the response of the cell lines may reflect their unique genetics. These results show the ability of the PCAIs to interfere with the MAPK pathway. As this signaling cascade plays a very important role in cell response as well as in the progression of cancers, therefore, therapeutics targeting this pathway have a great potential in the development of treatments not only for TNBC but also for other kinds of cancers. Citation Format: Jassy Mary Lazarte, Nada Tawfeeq, Yonghao Jin, Nazarius Lamango. Activation of MAP kinase pathway in breast cancer cell lines by polyisoprenylated cysteinyl amide inhibitors (PCAIs) causes cell death [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 2683.

Molecular characteristics and clinical outcomes of breast cancer with HRAS mutations.

561 Background: The RAS pathway regulates tumorigenesis and cell proliferation. HRAS is a RAS family member that activates via farnesylation. Indirectly targeting mutant HRAS with tipifarnib, a farnesyltransferase inhibitor (FTI), recently demonstrated efficacy in head and neck tumors. We aimed to investigate the molecular characteristics and clinical outcomes of HRAS mutations (HRASmut) for any potential role as a prognostic and therapeutic biomarker in breast cancer (BC). Methods: A total of 14,013 BC tissue samples had molecular profiling, including next generation DNA (592 Gene Panel, NextSeq, or WES, NovaSeq) or RNA sequencing (NovaSeq, WTS), and immunohistochemistry analyses, at Caris Life Sciences. MAP kinase (MAPK) activation and likelihood of a tumor’s response to anti-PD1 therapy were evaluated via MAPK Pathway Activity Score (MAPS) and interferon (IFN) score, respectively. Wilcoxon, Fisher’s exact, or Dunnett’s tests were used to determine statistical significance. Overall survival (OS) was calculated from date of tissue collection to insurance claims last contact using the Kaplan-Meier method. HRAS mutations (HRASmut) were compared to the general BC cohort (GC). Results: HRASmut were significantly enriched in older patients (median 69 vs 60 yrs; q&lt;.0001), and in primary compared to metastatic BC tumor samples (55.9% vs 41.9%, p&lt;.05). There were 70 total HRASmut (0.5%): Q61 was the most frequent (41.4%), followed by G12 (28.6%) and G13 (24.3%). Patients with Q61 HRASmut had significantly worse OS compared to GC (HR 1.86, 95% CI [1.10-3.13]; p&lt;0.05). HRASmut had significantly higher MPAS compared to GC (1.26, all; 1.31, Q61; 1.7, G12; -.39, GC, q&lt;.01). HRASmut were found in HR+/HER2- (22.6%) and TNBC (77.4%) tumors, but no HR-/HER2+ BC. TNBC samples with HRASmut displayed more PIK3CA (62.5% VS. 18.9%, q&lt;.05) but less TP53 mutations (50% vs 84.9%, q&lt;.05), higher expression of PD-L1 (41.2% vs 10.8%, p&lt;.05) and androgen receptor (AR, 45.8% vs 24.4%, p&lt;.05), and more frequent ARv7 fusions (20.7% vs 4.3%, p&lt;.05) compared to HR+/HER2- (Table 1). Q61 HRASmut had the highest MPAS (2.39 vs -0.28, p&lt;.01) in TNBC, whereas G12 HRASmut displayed the highest MPAS (2.01 VS -0.47, p&lt;.05) in HR+/HER2- BC. Conversely, Q61 had the lowest IFN score (-0.45 vs -0.3) in HR+/HER2- but the highest (-0.18 vs -0.3) in TNBC. Conclusions: HRASmut were mutually exclusive with HER2+ BC. The association of Q61 HRASmut with worse survival highlights the oncogenic role of these mutations and supports therapeutic investigation using FTI. PIK3CA was significantly co-mutated in HRASmut, highlighting a potential benefit of combining PIK3CA inhibitors with tipifarnib. Overall, HRASmut displayed a subtype-specific distinct genomic landscape and may represent a key therapeutic target in BC. [Table: see text]