Oncogene-induced Senescence Research Articles

Role of Senescence in Tumorigenesis and Anticancer Therapy.

Although the role of senescence in many physiological and pathological processes is becoming more identifiable, many aspects of senescence are still enigmatic. A special attention is paid to the role of this phenomenon in tumor development and therapy. This review mainly deals with a large spectrum of oncological issues, beginning with therapy-induced senescence and ending with oncogene-induced senescence. Moreover, the role of senescence in experimental approaches, such as primary cancer cell culture or reprogramming into stem cells, is also beginning to receive further consideration. Additional focus is made on senescence resulting from mitotic catastrophe processes triggered by events occurring during mitosis and jeopardizing chromosomal stability. It has to be also realized that based on recent findings, the basics of senescent cell property interpretation, such as irreversibility of proliferation blockade, can be undermined. It shows that the definition of senescence probably requires updating. Finally, the role of senescence is lately more understandable in the immune system, especially since senescence can diminish the effectiveness of the chimeric antigen receptor T-cell (CAR-T) therapy. In this review, we summarize the current knowledge regarding all these issues.

Zebrafish imaging reveals TP53 mutation switching oncogene-induced senescence from suppressor to driver in primary tumorigenesis

Most tumours are thought to arise through oncogenic cell generation followed by additional mutations. How a new oncogenic cell primes tumorigenesis by acquiring additional mutations remains unclear. We show that an additional TP53 mutation stimulates primary tumorigenesis by switching oncogene-induced senescence from a tumour suppressor to a driver. Zebrafish imaging reveals that a newly emerged oncogenic cell with the RasG12V mutation becomes senescent and is eliminated from the epithelia, which is prevented by adding a TP53 gain-of-function mutation (TP53R175H) into RasG12V cells. Surviving RasG12V-TP53R175H double-mutant cells senesce and secrete senescence-associated secretory phenotype (SASP)-related inflammatory molecules that convert neighbouring normal cells into SASP factor-secreting senescent cells, generating a heterogeneous tumour-like cell mass. We identify oncogenic cell behaviours that may control the initial human tumorigenesis step. Ras and TP53 mutations and cellular senescence are frequently detected in human tumours; similar switching may occur during the initial step of human tumorigenesis.

The Influence of Oncogenic RAS on Chemotherapy and Radiotherapy Resistance Through DNA Repair Pathways.

RAS oncogenes are chief tumorigenic drivers, and their mutation constitutes a universal predictor of poor outcome and treatment resistance. Despite more than 30 years of intensive research since the identification of the first RAS mutation, most attempts to therapeutically target RAS mutants have failed to reach the clinic. In fact, the first mutant RAS inhibitor, Sotorasib, was only approved by the FDA until 2021. However, since Sotorasib targets the KRAS G12C mutant with high specificity, relatively few patients will benefit from this therapy. On the other hand, indirect approaches to inhibit the RAS pathway have revealed very intricate cascades involving feedback loops impossible to overcome with currently available therapies. Some of these mechanisms play different roles along the multistep carcinogenic process. For instance, although mutant RAS increases replicative, metabolic and oxidative stress, adaptive responses alleviate these conditions to preserve cellular survival and avoid the onset of oncogene-induced senescence during tumorigenesis. The resulting rewiring of cellular mechanisms involves the DNA damage response and pathways associated with oxidative stress, which are co-opted by cancer cells to promote survival, proliferation, and chemo- and radioresistance. Nonetheless, these systems become so crucial to cancer cells that they can be exploited as specific tumor vulnerabilities. Here, we discuss key aspects of RAS biology and detail some of the mechanisms that mediate chemo- and radiotherapy resistance of mutant RAS cancers through the DNA repair pathways. We also discuss recent progress in therapeutic RAS targeting and propose future directions for the field.

Molecular Mechanisms of Alveolar Epithelial Stem Cell Senescence and Senescence-Associated Differentiation Disorders in Pulmonary Fibrosis.

Pulmonary senescence is accelerated by unresolved DNA damage response, underpinning susceptibility to pulmonary fibrosis. Recently it was reported that the SARS-Cov-2 viral infection induces acute pulmonary epithelial senescence followed by fibrosis, although the mechanism remains unclear. Here, we examine roles of alveolar epithelial stem cell senescence and senescence-associated differentiation disorders in pulmonary fibrosis, exploring the mechanisms mediating and preventing pulmonary fibrogenic crisis. Notably, the TGF-β signalling pathway mediates alveolar epithelial stem cell senescence by mechanisms involving suppression of the telomerase reverse transcriptase gene in pulmonary fibrosis. Alternatively, telomere uncapping caused by stress-induced telomeric shelterin protein TPP1 degradation mediates DNA damage response, pulmonary senescence and fibrosis. However, targeted intervention of cellular senescence disrupts pulmonary remodelling and fibrosis by clearing senescent cells using senolytics or preventing senescence using telomere dysfunction inhibitor (TELODIN). Studies indicate that the development of senescence-associated differentiation disorders is reprogrammable and reversible by inhibiting stem cell replicative senescence in pulmonary fibrosis, providing a framework for targeted intervention of the molecular mechanisms of alveolar stem cell senescence and pulmonary fibrosis. Abbreviations: DPS, developmental programmed senescence; IPF, idiopathic pulmonary fibrosis; OIS, oncogene-induced replicative senescence; SADD, senescence-associated differentiation disorder; SALI, senescence-associated low-grade inflammation; SIPS, stress-induced premature senescence; TERC, telomerase RNA component; TERT, telomerase reverse transcriptase; TIFs, telomere dysfunction-induced foci; TIS, therapy-induced senescence; VIS, virus-induced senescence.

Role of the Skin Microenvironment in Melanomagenesis: Epidermal Keratinocytes and Dermal Fibroblasts Promote BRAF Oncogene-Induced Senescence Escape in Melanocytes.

Simple SummaryMelanoma is a deadly skin cancer caused by the uncontrolled proliferation of melanocytes, a population of specialized cells that produce the skin pigment melanin. An aberrant proliferation of melanocytes is common, manifesting as new moles, and these lesions often remain benign. Only a small fraction of these aberrant melanocytes transition to melanoma (i.e., melanomagenesis). The factors that drive this transition are not fully understood. Recent studies have suggested that other cells—specifically, keratinocytes that make up the upper skin layers and fibroblasts, which are non-specialized cells within the deeper layers of the skin—also contribute to melanomagenesis. Here, employing microscale models that mimicked the skin microenvironment, we investigated the effect of crosstalk between melanocytes as well as keratinocytes and fibroblasts on melanomagenesis. Our findings show that keratinocyte- and fibroblast-derived factors can inhibit the mechanisms that prevent an uncontrolled melanocyte proliferation and contribute to melanomagenesis. Thus, targeting skin microenvironment-derived factors is a potential strategy to prevent melanomagenesis.BRAFV600E is the most common mutation driver in melanoma. This mutation is known to cause a brief burst of proliferation followed by growth arrest and senescence, which prevent an uncontrolled cell proliferation. This phenomenon is known as oncogene-induced senescence (OIS) and OIS escape is thought to lead to melanomagenesis. Much attention has been focused on the melanocyte-intrinsic mechanisms that contribute to senescence escape. Additional genetic events such as the loss of tumor suppressor PTEN and/or epigenetic changes that contribute to senescence escape have been described. However, the role of the skin microenvironment—specifically, the role of epidermal keratinocytes—on melanomagenesis is not fully understood. In this study, we employ a microfluidic platform to study the interaction between melanocytes expressing the BRAFV600E mutation as well as keratinocytes and dermal fibroblasts. We demonstrate that keratinocytes suppress senescence-related genes and promote the proliferation of transformed melanocytes. We also show that a keratinocyte-conditioned medium can alter the secretion of both pro- and anti-tumorigenic factors by transformed melanocytes. In addition, we show that melanocytes and keratinocytes from donors of white European and black African ancestry display different crosstalks; i.e., white keratinocytes appear to promote a more pro-tumorigenic phenotype compared with black keratinocytes. These data suggest that keratinocytes exert their influence on melanomagenesis both by suppressing senescence-related genes in melanocytes and by affecting the balance of the melanocyte-secreted factors that favor tumorigenesis.

Enterobacteria impair host p53 tumor suppressor activity through mRNA destabilization

Increasing evidence highlights the role of bacteria in the physiopathology of cancer. However, the underlying molecular mechanisms remains poorly understood. Several cancer-associated bacteria have been shown to produce toxins which interfere with the host defense against tumorigenesis. Here, we show that lipopolysaccharides from Klebsiella pneumoniae and other Enterobacteria strongly inhibit the host tumor suppressor p53 pathway through a novel mechanism of p53 regulation. We found that lipopolysaccharides destabilize TP53 mRNA through a TLR4-NF-κB-mediated inhibition of the RNA-binding factor Wig-1. Importantly, we show that K. pneumoniae disables two major tumor barriers, oncogene-induced DNA damage signaling and senescence, by impairing p53 transcriptional activity upon DNA damage and oncogenic stress. Furthermore, we found an inverse correlation between the levels of TLR4 and p53 mutation in colorectal tumors. Hence, our data suggest that the repression of p53 by Enterobacteria via TLR4 alleviates the selection pressure for p53 oncogenic mutations and shapes the genomic evolution of cancer.

Resistance to mutant KRASV12-induced senescence in an hTERT/Cdk4-immortalized normal human bronchial epithelial cell line

Mutant KRAS, the most frequently occurring (∼30%) driver oncogene in lung adenocarcinoma, induces normal epithelial cells to undergo senescence. This phenomenon, called “oncogene-induced senescence (OIS)”, prevents mutant KRAS-induced malignant transformation. We have previously reported that mutant KRASV12 induces OIS in a subset of normal human bronchial epithelial cell line immortalized with hTERT and Cdk4. Understanding the mechanism and efficacy of this important cancer prevention mechanism is a key knowledge gap. Therefore, this study investigates mutant KRASV12-induced OIS in upregulated telomerase combined with the p16/RB pathway inactivation in normal bronchial epithelial cells. The normal (non-transformed and non-tumorigenic) human bronchial epithelial cell line HBEC3 (also called “HBEC3KT”), immortalized with hTERT (“T”) and Cdk4 (“K”), was used in this study. HBEC3 that expressed mutant KRASV12 in a doxycycline-regulated manner was established (designated as HBEC3-RIN2). Controlled induction of mutant KRASV12 expression induced partial epithelial-to-mesenchymal transition in HBEC3-RIN2 cells, which was associated with upregulated expression of ZEB1 and SNAIL. Mutant KRASV12 caused the majority of HBEC3-RIN2 to undergo morphological changes; suggestive of senescence, which was associated with enhanced autophagic flux. Upon mutant KRASV12 expression, only a small HBEC3-RIN2 cell subset underwent senescence, as assessed by a senescence-associated β-galactosidase staining (SA-βG) method. Furthermore, mutant KRASV12 enhanced cell growth, evaluated by colorimetric proliferation assay, and liquid and soft agar colony formation assays, partially through increased phosphorylated AKT and ERK expression but did not affect cell division, or cell cycle status. Intriguingly, mutant KRASV12 reduced p53 protein expression but increased p21 protein expression by prolonging its half-life. These results indicate that an hTERT/Cdk4 -immortalized normal bronchial epithelial cell line is partially resistant to mutant KRASV12-induced senescence. This suggests that OIS does not efficiently suppress KRASV12-induced transformation in the context of the simultaneous occurrence of telomerase upregulation and inactivation of the p16/Rb pathway.

Loss of the transcription repressor ZHX3 induces senescence-associated gene expression and mitochondrial-nucleolar activation.

Cellular senescence is accompanied by metabolic and epigenomic remodeling, but the transcriptional mechanism of this process is unclear. Our previous RNA interference-based screen of chromatin factors found that lysine methyltransferases including SETD8 and NSD2 inhibited the senescence program in cultured fibroblasts. Here, we report that loss of the zinc finger and homeobox protein 3 (ZHX3), a ubiquitously expressed transcription repressor, induced senescence-associated gene expression and mitochondrial–nucleolar activation. Chromatin immunoprecipitation–sequencing analyses of growing cells revealed that ZHX3 was enriched at the transcription start sites of senescence-associated genes such as the cyclin-dependent kinase inhibitor (ARF-p16INK4a) gene and ribosomal RNA (rRNA) coding genes. ZHX3 expression was consistently downregulated in cells with replicative or oncogene-induced senescence. Mass spectrometry-based proteomics identified 28 proteins that interacted with ZHX3, including ATP citrate lyase and RNA metabolism proteins. Loss of ZHX3 or ZHX3-interaction partners by knockdown similarly induced the expression of p16INK4a and rRNA genes. Zhx3-knockout mice showed upregulation of p16INK4a in the testes, thymus and skeletal muscle tissues, together with relatively short survival periods in males. These data suggested that ZHX3 plays an essential role in transcriptional control to prevent cellular senescence.

Spatiotemporal expression of NRAS and occurrence of giant congenital melanocytic nevi.

The mechanism underlying giant congenital melanocytic nevus (GCMN) formation is not fully understood. According to recent research, NRAS gene mutation is the main driving factor in GCMN. Melanocytic precursor cells proliferate during the embryonic stage after acquiring NRAS mutations. However, why GCMN undergoes intense proliferation in the embryonic stage and then stops postnatally remains unknown. The current theory for this phenomenon is that the GCMN undergoes oncogene-induced senescence. However, there is not enough evidence to indicate that senescence induces growth arrest in GCMN. In this study, we hypothesized that the expression level of the NRAS gene changes dynamically during the development and differentiation of neural crest cells into melanocytes and that the NRAS expression level determines whether the cell proliferates or becomes quiescent.

MOB3A Bypasses BRAF and RAS Oncogene-Induced Senescence by Engaging the Hippo Pathway.

These results suggest that MOB3 targeting to re-engage the Hippo pathway, or direct targeting of YAP/TAZ, may be viable therapeutic strategies potential for RAS-pathway driven tumours.

Coordinated post-transcriptional control of oncogene-induced senescence by UNR/CSDE1.

Oncogene-induced senescence (OIS) is a form of stable cell-cycle arrest arising in response to oncogenic stimulation. OIS must be bypassed for transformation, but the mechanisms of OIS establishment and bypass remain poorly understood, especially at the post-transcriptional level. Here, we show that the RNA-binding protein UNR/CSDE1 enables OIS in primary mouse keratinocytes. Depletion of CSDE1 leads to senescence bypass, cell immortalization, and tumor formation, indicating that CSDE1 behaves as a tumor suppressor. Unbiased high-throughput analyses uncovered that CSDE1 promotes OIS by two independent molecular mechanisms: enhancement of the stability of senescence-associated secretory phenotype (SASP) factor mRNAs and repression of Ybx1 mRNA translation. Importantly, depletion of YBX1 from immortal keratinocytes rescues senescence and uncouples proliferation arrest from the SASP, revealing multilayered mechanisms exerted by CSDE1 to coordinate senescence. Our data highlight the relevance of post-transcriptional control in the regulation of senescence.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Elucidated pathogenesis and therapeutic prospects in Langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH) is characterized by immature dendritic cell proliferation, which is currently classified as an inflammatory myeloid neoplasm. Clinical features and outcomes vary from spontaneously regressing isolated bone disease to fatal liver, spleen, or hematopoietic system (risk organ) involvement-positive multisystem disease. LCH cells have the only mutation in the mitogen-activated protein kinase (MAPK) signaling pathway gene, represented by the BRAF V600E mutation, which is the driver mutation. The type of disease depends on the stage of hematopoietic cell differentiation at which the mutation occurs. LCH cells acquire anti-apoptosis and senescence-associated secretory phenotype by oncogene-induced senescence, with migration failure to lymph nodes. These cause LCH cell accumulation and various inflammatory cell recruitment in the lesion, resulting in severe inflammation. Tissue damage in LCH is due to this inflammation, not the LCH cell proliferation. Patients with a risk of organ involvement without the initial treatment response may be rescued by allogeneic hematopoietic stem cell transplantation after reducing the disease activity with MAPK inhibitors. Intravenous zoledronic acid and intrathecal cytarabine injections have been introduced into the ongoing clinical trial in Japan to reduce bone recurrence and prevent neurodegeneration as sequelae.

BRD7 Stabilizes P53 via Dephosphorylation of MDM2 to Inhibit Tumor Growth in Breast Cancer Harboring Wild-type P53.

Bromodomain-containing protein 7 (BRD7) was found to be down-expressed in nasopharyngeal carcinoma as well as breast cancer and to function as a potential tumor suppressor. BRD7 interacts with p53 and is required for p53-dependent oncogene-induced senescence. However, the mechanism how BRD7 functions as tumor suppressor roles in breast cancer remains unclear. MTT, colony formation assay, cell cycle, cell apoptosis, and tumorigenicity assays were performed to evaluate the biological functions of BRD7 in breast cancer cells in vitro and in vivo. Real-time PCR, western blot, luciferase reporter gene assays, and co-immunoprecipitation were used to examine the gene expression, transcription activation and protein-protein interaction. We reported that BRD7 effectively suppressed cell proliferation and tumor growth in vitro and in vivo. In addition, BRD7 increased p53 protein stability through ubiquitin-dependent proteasome pathway and regulated the expression of p53 downstream target genes by activating its transcriptional activity in breast cancers harboring wild-type p53. Mechanistically, BRD7 decreased phosphorylation and activation of MDM2 via inactivating its upstream kinase AKT depending on the bromodomain of BRD7, therefore BRD7 significantly reduced the amounts of phosphorylated MDM2 binding with p53 eventually decreasing ubiquitination level of p53. Furthermore, silencing the expression of p53 at least partly reversed the inhibition effect of BRD7 on cell proliferation and tumor growth in vitro and in vivo. Our studies identify that BRD7 stabilizes p53 by inhibiting the phosphorylation of MDM2 via AKT pathway dependent on its bromodomain to function as a tumor suppressor in breast cancer harboring wild-type p53.

Cytoplasmic innate immune sensing by the caspase-4 non-canonical inflammasome promotes cellular senescence.

Cytoplasmic recognition of microbial lipopolysaccharides (LPS) in human cells is elicited by the caspase-4 and caspase-5 noncanonical inflammasomes, which induce a form of inflammatory cell death termed pyroptosis. Here we show that LPS-mediated activation of caspase-4 also induces a stress response promoting cellular senescence, which is dependent on the caspase-4 substrate gasdermin-D and the tumor suppressor p53. Furthermore, we found that the caspase-4 noncanonical inflammasome is induced and assembled in response to oncogenic RAS signaling during oncogene-induced senescence (OIS). Moreover, targeting caspase-4 expression in OIS showed its critical role in the senescence-associated secretory phenotype and the cell cycle arrest induced in cellular senescence. Finally, we observed that caspase-4 induction occurs in vivo in mouse models of tumor suppression and ageing. Altogether, we are showing that cellular senescence is induced by cytoplasmic LPS recognition by the noncanonical inflammasome and that this pathway is conserved in the cellular response to oncogenic stress.

BRAFV600E induces reversible mitotic arrest in human melanocytes via microrna-mediated suppression of AURKB.

Benign melanocytic nevi frequently emerge when an acquired BRAFV600E mutation triggers unchecked proliferation and subsequent arrest in melanocytes. Recent observations have challenged the role of oncogene-induced senescence in melanocytic nevus formation, necessitating investigations into alternative mechanisms for the establishment and maintenance of proliferation arrest in nevi. We compared the transcriptomes of melanocytes from healthy human skin, nevi, and melanomas arising from nevi and identified a set of microRNAs as highly expressed nevus-enriched transcripts. Two of these microRNAs-MIR211-5p and MIR328-3p-induced mitotic failure, genome duplication, and proliferation arrest in human melanocytes through convergent targeting of AURKB. We demonstrate that BRAFV600E induces a similar proliferation arrest in primary human melanocytes that is both reversible and conditional. Specifically, BRAFV600E expression stimulates either arrest or proliferation depending on the differentiation state of the melanocyte. We report genome duplication in human melanocytic nevi, reciprocal expression of AURKB and microRNAs in nevi and melanomas, and rescue of arrested human nevus cells with AURKB expression. Taken together, our data describe an alternative molecular mechanism for melanocytic nevus formation that is congruent with both experimental and clinical observations.

Cellular Senescence in Adrenocortical Biology and Its Disorders.

Cellular senescence is considered a physiological process along with aging and has recently been reported to be involved in the pathogenesis of many age-related disorders. Cellular senescence was first found in human fibroblasts and gradually explored in many other organs, including endocrine organs. The adrenal cortex is essential for the maintenance of blood volume, carbohydrate metabolism, reaction to stress and the development of sexual characteristics. Recently, the adrenal cortex was reported to harbor some obvious age-dependent features. For instance, the circulating levels of aldosterone and adrenal androgen gradually descend, whereas those of cortisol increase with aging. The detailed mechanisms have remained unknown, but cellular senescence was considered to play an essential role in age-related changes of the adrenal cortex. Recent studies have demonstrated that the senescent phenotype of zona glomerulosa (ZG) acts in association with reduced aldosterone production in both physiological and pathological aldosterone-producing cells, whereas senescent cortical-producing cells seemed not to have a suppressed cortisol-producing ability. In addition, accumulated lipofuscin formation, telomere shortening and cellular atrophy in zona reticularis cells during aging may account for the age-dependent decline in adrenal androgen levels. In adrenocortical disorders, including both aldosterone-producing adenoma (APA) and cortisol-producing adenoma (CPA), different cellular subtypes of tumor cells presented divergent senescent phenotypes, whereby compact cells in both APA and CPA harbored more senescent phenotypes than clear cells. Autonomous cortisol production from CPA reinforced a local cellular senescence that was more severe than that in APA. Adrenocortical carcinoma (ACC) was also reported to harbor oncogene-induced senescence, which compensatorily follows carcinogenesis and tumor progress. Adrenocortical steroids can induce not only a local senescence but also a periphery senescence in many other tissues. Therefore, herein, we systemically review the recent advances related to cellular senescence in adrenocortical biology and its associated disorders.

A recurrent chromosomal inversion suffices for driving escape from oncogene-induced senescence via subTAD reorganization

Oncogene-induced senescence (OIS) is an inherent and important tumor suppressor mechanism. However, if not removed timely via immune surveillance, senescent cells also have detrimental effects. Although this has mostly been attributed to the senescence-associated secretory phenotype (SASP) of these cells, we recently proposed that "escape" from the senescent state is another unfavorable outcome. The mechanism underlying this phenomenon remains elusive. Here, we exploit genomic and functional data from a prototypical human epithelial cell model carrying an inducible CDC6 oncogene to identify an early-acquired recurrent chromosomal inversion that harbors a locus encoding the circadian transcription factor BHLHE40. This inversion alone suffices for BHLHE40 activation upon CDC6 induction and driving cell cycle re-entry of senescent cells, and malignant transformation. Ectopic overexpression of BHLHE40 prevented induction of CDC6-triggered senescence. We provide strong evidence in support of replication stress-induced genomic instability being a causative factor underlying "escape" from oncogene-induced senescence.

MZF1 mediates oncogene-induced senescence by promoting the transcription of p16INK4A.

Oncogene induced senescence is a tumor suppressing defense mechanism, in which the cell cycle-dependent protein kinase (CDK) inhibitor p16INK4A (encoded by the CDKN2A gene) plays a key role. We previously reported that a transcriptional co-activator chromodomain helicase DNA binding protein 7 (CHD7) mediates oncogenic ras-induced senescence by inducing transcription of the p16INK4A gene. In the current study, we identified myeloid zinc finger 1 (MZF1) as the transcriptional factor that recruits CHD7 to the p16INK4A promoter, where it mediates oncogenic ras-induced p16INK4A transcription and senescence through CHD7, in primary human cells from multiple origins. Moreover, the expression of MZF1 is induced by oncogenic ras in senescent cells through the c-Jun and Ets1 transcriptional factors upon their activation by the Ras-Raf-1-MEK-ERK signaling pathway. In non-small cell lung cancer (NSCLC) and pancreatic adenocarcinoma (PAAD) where activating ras mutations occur frequently, reduced MZF1 expression is observed in tumors, as compared to corresponding normal tissues, and correlates with poor patient survival. Analysis of single cell RNA-sequencing data from PAAD patients revealed that among the tumor cells with normal RB expression levels, those with reduced levels of MZF1 are more likely to express lower p16INK4A levels. These findings have identified novel signaling components in the pathway that mediates induction of the p16INK4A tumor suppressor and the senescence response, and suggested that MZF1 is a potential tumor suppressor in at least some cancer types, the loss of which contributes to inactivation of the p16INK4A/RB pathway and disruption of senescence in tumor cells with intact RB.

ERK2 Substrate Binding Domains Perform Opposing Roles in Pathogenesis of a JAK2V617F-Driven Myeloproliferative Neoplasm

The interaction between ERK2 and its substrates is critically mediated by two domains, the common docking (CD) D-domain and DEF-binding pocket (DBP) domain. Previous studies have suggested that ERK2 is not only necessary to drive hematopoietic and myelo-erythroid development, but it is also important for the pathogenesis of hematological cancers, as revealed by recurrent ERK2 somatic mutations in many types of lymphoma and leukemia. Here we show that the activation of ERK2 in JAK2V617F-driven myeloproliferative neoplasm (MPN) enhance polycythemia vera (PV) progression from erythrocytosis to myelofibrosis when ERK2-DBP binding capacity is disabled. Conversely, targeting the ERK2 D-domain preserves ERK2 catalytic function while reducing outgrowth and proliferation of human and murine MPN cell lines.To determine whether ERK2 binding domains play a role in the pathogenesis of JAK2V617F-driven MPN, we generated an ERK2-Y261A (Erk2 Y261A) mutant knockin mouse model in which the ERK2-DBP domain was inactivated, but ERK2 kinase activity and D-domain function was preserved. We observed splenomegaly in Erk2 Y261A/Y261A, characterized by a 10-fold expansion of CD44+/Ter119+ primitive erythroblasts and Ter119+CD44+/ FSC hi immature erythroid progenitors. We then transplanted Jak2 V617F-expressing ERK2 wildtype (WT), knockout (KO) or DBP hematopoietic progenitors (HSPCs) into irradiated, immunodeficient mice (Rag2 −/−Il2rg −/−) and observed disease resembling human PV, with elevated RBC counts, hematocrit, and hemoglobin levels. ERK2-DBP mutant recipients exhibited mild but sustained erythrocytosis with a prevalence of circulating GFP+ monocytes and neutrophils. At 12-weeks post-transplantation, ERK2-DBP mutant recipients had significant splenic burden and decreased bone marrow cellularity relative to WT and KO recipients. Reticulin staining confirmed progression towards myelofibrosis with interstitial infiltration in both the BM and the spleen of ERK2 DBP mutant recipients.To understand the molecular basis of ERK-DBP domain in JAK2V617F-driven MPN, we performed in vitro colony forming unit assay to assess the clonogenic potential of JAK2V617F-expressing HSPCs in cytokine supplemented-methylcellulose supporting myeloid or erythroid progenitor development. Ectopic JAK2V617F expression markedly reduced colony formation by both ERK2 wildtype and knockout HSPCs. On the other hand, JAK2V617F expression did not reduce colony formation by ERK2-DBP HSPCs. These differences were not evident in methylcellulose cultures that supported erythroid development, suggesting that disabling ERK-DBP binding capacity primarily affects the myeloid lineage. Furthermore, we found that JAK2V617F increased senescence associated β-galactosidase (SA-βGa) activity in ERK2-WT HSPCs. SA-βGal activity was modestly affected by ERK deficiency but was strongly attenuated by the ERK2-DBP mutation, supporting the notion that ERK2-DBP domain promotes oncogene-induced senescence (OIS). Previously, we identified transcription factor, Early growth response 1 (Egr1) which interacts with ERK2-DBP domain and plays a critical role as a tumor suppressor in myeloid neoplasms. We observed that JAK2V617F expression failed to suppress colony formation and senescence by Egr1-deficient HSPCs. The ability of EGR1 to restore these effects is abrogated by inactivating the DEF motif (EGR1-Y252A). Together, this data demonstrates that the interaction between ERK2-DBP and Egr1 is required for JAK2V617F-mediated colony suppression and senescence induction.Finally, to determine whether ERK-D domain acts to promote progression by targeting substrates distinct from those that bind to the DBP domain, we developed an inhibitor (#76) of the ERK-D domain. Indeed, 76 spared ERK phosphorylation and EGR1 induction but impaired RSK phosphorylation, a critical D-domain substrate involved in cancer progression. Moreover, 76 suppressed colony formation by JAK2V61F-expressing HSPCs akin to the active site MEK/ERK inhibitor, U0126. By altering the 4-ethoxybenylidene moiety of 76, we were able to enhance anti-proliferative effects of the parent compound against human MPN cell lines, SET-2, UKE-1, and Ku812. Our findings identify ERK2-domain specific roles in the pathogenesis of JAK2V617F driven MPN and supports a novel therapeutic approach to targeting JAK2 and MAPK dependent MPN. DisclosuresLevine: Celgene: Research Funding; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Lilly: Honoraria; Janssen: Consultancy; Astellas: Consultancy; Gilead: Honoraria; Prelude: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy; Roche: Honoraria, Research Funding; Imago: Membership on an entity's Board of Directors or advisory committees. Wiest: Janssen Pharmaceuticals: Research Funding.