Constitutive Activation Of ERK Research Articles

Sustained ERK signaling promotes G2 cell cycle exit and primes cells for whole-genome duplication.

Whole-genome duplication (WGD) is a frequent event in cancer evolution that fuels chromosomal instability. WGD can result from mitotic errors or endoreduplication, yet the molecular mechanisms that drive WGD remain unclear. Here, we use live single-cell analysis to characterize cell-cycle dynamics upon aberrant Ras-ERK signaling. We find that sustained ERK signaling in human cells leads to reactivation of the APC/C in G2, resulting in tetraploid G0-like cells that are primed for WGD. This process is independent of DNA damage or p53 but dependent on p21. Transcriptomics analysis and live-cell imaging showed that constitutive ERK activity promotes p21 expression, which is necessary and sufficient to inhibit CDK activity and which prematurely activates the anaphase-promoting complex (APC/C). Finally, either loss of p53 or reduced ERK signaling allowed for endoreduplication, completing a WGD event. Thus, sustained ERK signaling-induced G2 cell cycle exit represents an alternative path to WGD.

Dynamic Recruitment of Inhibitory Complexes Controls Oncogenic Signaling in B-Cell Malignancies

Background and Significance: Initiation of B-cell receptor (BCR) signaling, and subsequent antigen-encounter in germinal centers represent milestones of B-lymphocyte development that are both marked by sharp increases of CD25 surface-expression. Oncogenic signaling in B-cell leukemia (B-ALL) and lymphoma also induced CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells, the significance of its expression on B-cells was unclear. High expression levels on the surface of B-ALL cells are associated with poor clinical outcomes in pediatric (COG P9906) and adult (ECOG E2993) B-ALL cohorts. In DLBCL, aggressive disease is associated with CD25-inactivation by shedding and high serum levels of soluble CD25. Results: Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells assembled an inhibitory complex including PKCδ, immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptors and inhibitory phosphatases for feedback control of BCR-signaling or its oncogenic mimics. Recapitulating phenotypes of genetic ablation of PKCδ and phosphatases, conditional CD25-deletion decimated early B-cell subsets but expanded mature B-cell populations and induced autoimmunity. In B-cell malignancies arising from early stages of B-cell development (e.g. B-ALL) CD25-loss induced hyperactivation of oncogenic signaling followed by cell death. In contrast, genetic deletion of CD25 in models for mature B-cell lymphoma accelerated proliferation of malignant cells. Biochemical and interactome studies revealed a critical role of CD25 in feedback regulation of oncogenic signaling in B-cell malignancies: Signaling from BCR-ABL1 and RAS-oncogenes in B-ALL as well as oncogenic BCR-signaling in DLBCL induced PKCδ-mediated phosphorylation of CD25 on its cytoplasmic tail (S 268). Importantly, CD25-phosphorylation stabilized its expression at the cell membrane and opposed both inactivation by CD25-shedding and internalization. Moreover CD25-S 268 tail-phosphorylation enabled CD25 to recruit inhibitory receptors, including CD22, that bear ITIM-motifs for the activation of inhibitory phosphatases (e.g. SHP1, Figure 1). Thereby, the positively charged cytoplasmic tail of CD25 engages negatively charged residues in the cytoplasmic tails of the BCR signaling chain CD79B and the inhibitory CD22-ITIM. Molecular dynamics simulations revealed that the ternary complex between CD25, CD79B and CD22 enables recruitment and activation of the phosphatase SHP1 within reach of its substrate, the BCR signaling chain CD79B. Thereby, the SH2-domain of SHP1 engages the CD22-ITIM motif, while its phosphatase domain makes contact with the CD79B-ITAM to dephosphorylate and terminate BCR-signaling. This complex, while engaging and activating SHP1, is held together by CD25 ( Figure 1). To test functional consequences of destabilization of this complex, we generated a knockin mouse model carrying a single point mutation (S268A) in the cytoplasmic tail of CD25. The CD25 S268A knockin mutation abolished recruitment and activation of SHP1. In addition, the S268A-mutant failed to assemble CD22 and CD79B in complex with SHP1 to limit duration and strength of BCR-signaling. Loss of phosphatase-function and failure to terminate oncogenic signaling resulted in constitutive activation of ERK and NF-κB, autonomous Ca 2+-oscillations. Hyperactivation of oncogenic signaling resulted in exhaustion and cell death in B-ALL cells, as opposed to excessive proliferation and acceleration of disease in mature B-cell lymphoma. Conclusions: While CD25 has an established function in IL2 signal transduction in T- and NK-cells, these findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to control oncogenic signaling in B-cell malignancies and provides an explanation for the common feature of CD25 shedding and high serum levels of functionally inactive soluble CD25 in aggressive B-cell lymphomas.

Immunoglobulin Light Chains Control Permissiveness to Malignant B-Cell Transformation By RAS-Pathway Lesions

Background: RAS-pathway activating mutations (e.g. NRAS, KRAS, BRAF) cause constitutive ERK-activation which can lead to divergent outcomes. ERK-activation not only drives early stages of pre-B cell differentiation (Yasuda et al., 2008) but also induces negative selection and cell death to eliminate aberrantly activated B-cells at later stages of development (Limnander et al., 2011). Germline mutations in this pathway cause lymphoproliferative disorder and autoimmunity but not leukemia. Furthermore, activating lesions are very common in B-cell malignancies derived from the earliest (e.g. B-ALL) and the latest (e.g. multiple myeloma) stages of B-cell development, but they are rarely found in B-cell receptor (BCR)-expressing mature B-cell derived tumors ( Figure 1). These findings are consistent with previous observations that transgenic mouse models for RAS-oncogene were able to transform BCR-negative B-cell precursors to develop B-cell acute lymphoblastic leukemia (Chan et al., 2020). In contrast, transgenic expression of these oncogenes fails to transform BCR-positive mature B-cells (Mullins et al., 2013; Chung et al., 2014). Signals from the BCR, consisting of immunoglobulin (Ig) heavy chains (μ HC) and conventional light chains (kappa LC or lambda LC), and its precursor the pre-BCR, consisting of μ HC and surrogate light chains (SLC; λ5 and VpreB), regulate the development and function of B-cells. Here, we investigated whether structural elements of the (pre-) BCR determine the outcome of oncogenic RAS activation and how oncogenic RAS in B-ALL avoids cell death to promote leukemogenesis. Results: We first studied the impact of oncogenic RAS-activation in BCR-positive mantle cell lymphoma cells. Expression of NRAS G12D in BCR-positive mantle cell lymphoma (JeKo-1) cells transiently reduced the surface expression of μ HC and eventually resulted in depletion of cells from culture in growth competition assays. We then examined whether CRISPR/Cas9-based genetic ablation of BCR components will enable permissiveness to oncogenic RAS signaling. Despite loss of μ HC function, oncogenic RAS caused depletion of JeKo-1 cells from culture. In contrast, this effect was partially reversed by genetic ablation of the constant region of immunoglobulin kappa LC ( IGKC). We next examined whether reconstitution of surrogate light chain (SLC) components of the pre-BCR will confer permissiveness to oncogenic RAS signaling in JeKo-1 cells. We engineered JeKo-1 cells to express Dox-inducible expression of λ5 (Igll1) and VpreB. In the presence of kappa LC, overexpression of SLC alone did not reverse the deleterious impact of oncogenic RAS expression. Notably, reconstitution of SLC components following genetic deletion of IGKC amplified the effect of IGKC-deletion alone and largely rescued cells from oncogenic RAS-induced survival disadvantage. Taken together, our results showed that immunoglobulin light chains control the consequences of oncogenic RAS activation in B-cells - while SLC of the pre-BCR plays a productive role in enabling oncogenic RAS-signaling, conventional light chain of the BCR plays a counterproductive role. Supporting this scenario, we found that SLC enabled oncogenic RAS to massively induce the proto-oncogene Bcl6 at the expense of the tumor suppressor Prdm1 to avoid cell death, promoting RAS-driven transformation of B-cell precursors. Upon genetic ablation of λ5, upregulation of Bcl6 was abrogated while Prdm1 was de-repressed. Consequently, RAS-driven malignant transformation of B-cell precursors was subverted. Reflecting the critical role of SLC in enabling RAS-BCL6 signaling, mature B-cells, which express BCR and lack SLC, failed to induce Bcl6 upon oncogenic RAS-activation, leading to upregulation of Prdm1. This observation may explain why persistent activation of the RAS-pathway in mature B-cells can lead to negative selection and cell death (Limnander et al., 2011). Conclusions: Our results demonstrated that immunoglobulin light chains determine permissiveness to RAS-driven malignant transformation of B-cells. Expression of SLC enables RAS-BCL6 activation, allowing B-cell precursors to avoid PRDM1-mediated tumor suppression to promote leukemogenesis. In contrast, conventional LC opposes oncogenic RAS signaling, providing a mechanistic basis of the low frequency of RAS-pathway activating mutations in BCR-expressing mature B-cell lymphomas.

Abstract 1431: Sprouty 2 expression attenuates proliferation in nras-driven acute myeloid leukemia cell lines

Abstract Oncogenic mutations in the NRAS gene are found in many cancers including a subset of acute myeloid leukemias (AML). Established AML cell lines exhibiting hyperactive signaling through the RAS-RAF-MEK-ERK pathway show constitutive ERK activation generating a transcriptome that mediates escape from programmed cell death and continuous proliferation. It has been established that exposure of many AML cell lines to phorbol esters such as phorbol-12-myristate-13-acetate (PMA) mediates cell cycle arrest, myeloid differentiation, and apoptosis. This effect seems counterintuitive given that PMA irreversibly activates protein kinase C (PKC), which enhances ERK activity leading to transcriptional upregulation of target genes controlled by ERK activated transcription factors, many of which promote proliferation. Here we define a set of PMA response genes in AML cell lines driven by oncogenic NRAS that encode negative regulators of RAS-RAF-MEK-ERK signaling, including SPRY2, RASA1, DUSP 5, and DUSP6. Additionally, we show that overexpression of SPRY2 alone attenuates proliferation by inhibiting ERK activation and altering the AML cell transcriptome through the activation of genes encoding members of the AP-1 transcription factor family, FOS, JUNB, and JUND, and the cyclin-CDK inhibitors CDKN1A and CDKN2B. We hypothesize that oncogenic NRAS signaling is overridden by PMA treatment through the activation of ERK target genes that ultimately inhibit the MAPK-ERK pathway, such as SPRY2. Finally, we show that SPRY 2 expression in AML patient samples represents a survival prognostic indicator where higher expression levels correlate with improved overall survival. Citation Format: Michael Roberts, Sher Bahadur, Coltin Albitz, Jae Chung, Samantha Garcia, Cuong Nguyen, Sophia Kovatsis. Sprouty 2 expression attenuates proliferation in nras-driven acute myeloid leukemia cell lines [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 1431.

Structural Elements of the BCR Determine Permissiveness to Oncogenic RAS-Driven B-Cell Transformation

Background: RAS-pathway activating mutations (e.g. NRAS, KRAS, BRAF) invariably promote constitutive ERK-activation and are oncogenic drivers in B-cell acute lymphoblastic leukemia (B-ALL; Irving et al., 2014). Conversely, persistent ERK-activation can induce plasma cell differentiation (Yasuda et al., 2011), negative B-cell selection and cell death (Limnander et al., 2011) during B-cell development. In RAS-associated autoimmune leukoproliferative disorder (RALD), RAS-activating germline mutations cause lymphoproliferative disorder but not leukemia. The molecular determinants driving these divergent outcomes of RAS-activation have yet to be characterized. For these reasons, we here examined the mechanisms that enable permissiveness to RAS-driven transformation of B-cells. Results: Studying RAS-pathway activating mutations, we found that these genetic lesions are very common in B-cell malignancies derived from the earliest (e.g. B-ALL) and the latest (e.g. multiple myeloma) stages of B-cell development. In contrast, RAS-pathway lesions are exceedingly rare in B-cell receptor (BCR)-expressing B-cell malignancies, including mantle cell lymphoma, chronic lymphocytic leukemia, follicular lymphoma, Burkitt's lymphoma and diffuse large B-cell lymphoma (Figure 1). We also studied the consequence of oncogenic RAS-activation in mature B-cells, which express BCR. Consistent with previous findings that ERK-activation results in negative B-cell selection and cell death of B-cells (Limnander al., 2011), we found that expression of NRASG12D in mature B-cells resulted in upregulation of Prdm1, a tumor suppressor and mediator of negative B-cell selection and cell death (Hug et al., 2014; Setz et al., 2018). Altogether, our results suggest that mature B-cells are not permissive to RAS-driven transformation and prompted us to investigate whether expression of BCR counteracts constitutive ERK-activation resulting from oncogenic RAS signaling in B-cells. Reflecting incompatibility between oncogenic RAS-ERK activation and BCR-expressing malignant B-cells, expression of NRASG12D in BCR-positive mantle cell lymphoma cells transiently decreased surface expression of BCR but eventually led to depletion of cells from culture in growth competition assays. The non-receptor protein tyrosine kinase SYK is a key mediator of BCR signaling. Notably, treatment with small molecule inhibitor of SYK (entospletinib) largely reversed cell death caused by oncogenic RAS-signaling. Taken together, our results suggest that expression of an active BCR prevents permissiveness to constitutive ERK-activation. BCR consists of µ heavy chain (µHC) and conventional light chains (LCs; either kappa LC or lambda LC). Given that expression of a functional BCR opposes constitutive ERK-activation, this raised the question of whether structural elements of BCR are involved in determining outcome of RAS-activation. To this end, we performed CRISPR/Cas9-based gene editing to disrupt the constant region of immunoglobulin heavy chain (IGHM) or the constant region of the immunoglobulin kappa LC (IGKC) in kappa LC-expressing mantle cell lymphoma cells. While CRISPR-mediated deletion of IGHM reduced surface expression of IgM (μHC), this did not rescue cells from call death caused by oncogenic RAS-ERK activation. On the contrary, genetic ablation of IGKC enabled permissiveness to oncogenic RAS-signaling and prevented cell death resulting from constitutive ERK-activation. Hence, these findings showed that expression of kappa LC opposes constitutive RAS-ERK signaling in B-cell lymphoma cells. Conclusions: Surface expression of BCR is conserved in most mature B-cell lymphomas. Recent studies suggest that constitutive RAS-ERK signaling can override BCR requirement in B-cell lymphoma and that B-cell lymphoma cells acquiring RAS mutations may lose selective pressure to express a functional BCR (Varano et al., 2017). Here, our results demonstrated that conventional LCs of the BCR determine permissiveness to RAS-driven malignant transformation of B-cells. Conventional LCs were previously shown to inhibit the autonomous signaling capacity of BCR (Meixlsperger et al., 2007). We here showed that conventional LCs of BCR counteract oncogenic RAS signaling, providing a mechanistic basis of the low frequency of RAS-pathway activating mutations observed in BCR-expressing B-cell malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Potential Drivers of Acquired Resistance to Idelalisib in CLL Patients

Idelalisib is an isoform-specific PI3-kinase-δ inhibitor approved for the treatment of chronic lymphocytic leukemia (CLL). We seek to understand the biologic basis of the variability in depth of response among patients. We have previously reported that intrinsic resistance to idelalisib is associated with RAS/RAF/MAP2K1 mutations and constitutive ERK activation [Blood 2021; 138: 44]. Unlike these non-responder patients, some patients with initial response will acquire resistance to therapy over time, but molecular mechanisms of this acquired resistance to idelalisib remain elusive. To identify somatic mutations that fuel acquired resistance, we performed whole exome sequencing of serial PBMC samples from three CLL patients who responded to idelalisib, but later acquired resistance (average 4.1 yrs from start). We observed that all three patients acquired distinct somatic mutations during idelalisib treatment. Patient 1 harbored TP53 and BIRC3 at baseline, but at progression (5.3 yrs later) had acquired somatic mutations in RAF1, PDIA3, RB1, and ATM. Patient 2 had baseline mutations in SF3B1, BRCA2, and HIST1HIE but acquired additional mutations in PIK3R1 and IKZF3 at progression (4.4 yrs later). Patient 3 carried somatic mutations in TP53 and SF3B1 at baseline and acquired somatic mutations in DDX3X, NOTCH1, FBXW7, CD79B, NFATC1 and NFATC3 at progression (2.7 yrs later). To identify novel drivers of resistance and validate our findings from the initial three CLL patients, we sequenced progression samples from eight additional CLL patients following resistance to idelalisib treatment. In addition to patient 1, we detected mutations in BIRC3 in two additional CLL cases, and mutations in KRASand NRAS (both of which are known to aberrantly activate the MAPK pathway) in two and one CLL patients, respectively, separate from the cases with BIRC3 mutation. In addition to the PIK3R1 acquired mutation in patient 2, we also identified mutations in other PI3K pathway-related genes, such as PIK3CA, PIK3CG, andPLCG2. Since we only sequenced progression samples from these 8 patients, we cannot confirm whether the mutations were acquired or were present in the baseline samples. Overall, these results suggest that mutations in the RAS/RAF/MAP2K1 pathway, as well as in the PI3K pathway itself, and BIRC3 (NF-kB pathway), are associated with PI3K inhibitor resistance. To identify expression programs that may lead to idelalisib resistance, we performed comparative transcriptomic profiling of responding and resistant time point samples from the three initially studied CLL patients (patient 1, 2 and 3). In this analysis, we detected 214 differentially expressed genes with adjusted p-value < 0.05 and fold change of 1.5. GSEA analysis revealed enrichment of cancer related cell signaling pathways, most notably an enrichment for the MAPK pathway (FDR q-value- 0.04). We found that AICDA, LTK, and MAPK12 are overexpressed in resistant samples from the three original CLL patients with acquired resistance as compared to the matched responding time points. In a separate cohort of frontline patients treated with idelalisib, we also found that these same 3 genes were consistently overexpressed before, during and after treatment in early progressors after idelalisib discontinuation. Higher expression of AICDA in progression samples may drive resistance by inducing somatic mutations and/or altering the epigenome of the tumor cells in CLL patients. LTK receptor tyrosine kinase activates PI3K and MAPK pathways in hematopoietic cells. Previously we have reported that MAPK pathway activation plays a key role in primary resistance to idelalisib in CLL, hence overexpression of LTK is a possible mechanism of MAPK activation. We checked the phosphorylation/activation level of AKT and ERK1/2 at the responding and progression time points in the three original CLL patients with acquired resistance and observed that pERK levels are inhibited by idelalisib at baseline, but not at progression, in 2 patients (patient 1 and 2). In conclusion, we have identified potential drivers of acquired resistance to idelalisib in CLL patients, including MAPK pathway activation as in our prior study, and we continue with ongoing work evaluating the potential role of PI3K pathway mutations, BIRC3, AICDA and LTK in treatment resistance, since insights from these studies will help us guide therapy for CLL patients with refractory disease.

BCL6-Mediated Escape from Negative Selection Enables RAS-Driven B-Cell Transformation

Background: In B-cell acute lymphoblastic leukemia (B-ALL), RAS-pathway activating mutations (e.g. NRAS, KRAS, BRAF) are found in ~40% of cases (Irving et al., 2014; Jerchel et al., 2018; Zhang et al., 2011). Evidence suggests that RAS-pathway mutations in B-ALL subclones are selected for during therapy and are associated with relapse, which has a poor prognosis and remains one of the leading causes of death in children with malignancy (Irving et al., 2014; Jerchel et al., 2018). Beyond its role as a driver of B-ALL, ERK-activation can induce expression of PRDM1, which functions as a tumor suppressor and triggers negative B-cell selection and cell death (Yasuda et al., 2011; Hug et al., 2014; Setz et al., 2018). During B-cell development, PRDM1 and the proto-oncogene BCL6 are reciprocal antagonists (Shaffer et al., 2002). Here, we examined the mechanism by which oncogenic RAS-ERK signaling in B-ALL avoids negative selection and cell death to promote leukemogenesis. Results: Mimicking survival signals from pre-B cell receptor (pre-BCR), constitutive Erk activation upon oncogenic RAS (NRASG12D) expression in mouse B-cell precursors massively induced the mRNA (~390-fold) and protein (~50-fold) levels of the proto-oncogene Bcl6. Highlighting an essential role of Erk-signaling in upregulation of Bcl6 expression downstream of oncogenic RAS, pharmacological Erk-inhibition markedly reduced NRASG12D-mediated Bcl6 induction. Both the pre-BCR linker molecule Blnk and the surrogate light chains (SLCs) of the pre-BCR are required for Erk-mediated induction of Bcl6. Genetic ablation of Blnk or the SLC component λ5 abrogated RAS-mediated Bcl6-induction. Hence, oncogenic expression of Bcl6 in RAS-driven B-ALL cells depended on structural elements of the pre-BCR. Additionally, ERK-activation in patient-derived B-ALL cells had the same effect on BCL6 expression. Pharmacological reactivation of ERK (BCI-215) markedly induced BCL6 expression, whereas small molecule inhibition of ERK-activity (trametinib) reduced BCL6 levels in patient-derived B-ALL cells. Consistent with BCL6-transactivation downstream of ERK-activation, ChIP-seq analysis revealed binding of ERK-dependent transcription factors (CREB1, ELK1, EGR1, JUND and C-JUN) to the BCL6 promoter in human B cells. Altogether, our findings illuminated a critical role of ERK-signaling in positive regulation of BCL6 expression. Using a genetic model for Cre-mediated deletion of Bcl6 in NRASG12D B-ALL cells, we found that Bcl6 was required for leukemia-initiation in transplant recipient mice (P=0.007). Patient-derived RAS-driven B-ALL cells were highly sensitive to pharmacological inhibition of BCL6 using peptide (RI-BPI) and small molecule (FX1) inhibitors. Furthermore, pharmacological inhibition of BCL6 delayed onset of fatal disease and prolonged survival of transplant recipient mice bearing patient-derived RAS-driven B-ALL cells (P=0.009). Taken together, our findings demonstrated that suppressing BCL6 function is a promising option for targeted therapeutics in RAS-driven B-ALL. Mechanistically, genetic ablation or pharmacological inhibition of BCL6 increased PRDM1 expression. Gene expression analysis revealed that ectopic expression of Prdm1 in B-cell precursors promoted gene expression programs of apoptosis and cell cycle arrest (GSE111692). While Bcl6-deletion resulted in cell death of B-ALL cells, this was largely reversed by shRNA-mediated silencing of Prdm1. Altogether, our results showed that compromised leukemogenesis was a result of aberrant PRDM1 expression in BCL6-deficient RAS-driven B-ALL cells. Conclusions: We previously investigated targeted engagement of negative selection in B-ALL and showed that hyperactivation of SYK or AKT functionally mimics autoreactive pre-BCR signaling and results in negative selection and cell death (Chen et al., 2015; Shojaee et al., 2016). Here, we reinforced this concept by providing a mechanistic basis on how negative selection is downregulated in RAS-driven B-ALL and how it can be reactivated for therapeutic benefit - namely, BCL6 curbs ERK-mediated induction of PRDM1 expression to promote leukemogenesis and this mechanism can be exploited as synthetic lethality in the treatment of this disease.

Structural Basis of Feedback Control of Oncogenic Signaling in B-Lymphoid Malignancies

Introduction: Multiple structural elements are known that recruit kinases to sites of active signal transduction (e.g. lipid rafts) within the plasma membrane. For instance, pleckstrin homology (PH) domains and the conserved intracellular loop (CIL) motifs recently discovered by us (Lee et al., Nature 2021) direct kinase molecules (Src, BTK, AKT and PI3K) to large signaling clusters in lipid rafts to amplify normal or oncogenic B-cell signaling. While concepts of site-specific recruitment of kinases to initiate signaling are well established, little is known about spatial control of inhibitory phosphatases and how they are directed to sites of maximal signaling strength.Results: Here we discovered the cytoplasmic tail of CD25 as an essential structural element for site-specific recruitment of inhibitory phosphatases. CD25-mediated shuttling of inhibitory phosphatases balances oncogenic signaling strength and is essential for cell-survival in B-cell malignancies, including B-ALL, CLL and mantle cell lymphoma. This was unexpected because CD25 (IL2RA) is known as one of three chains of the Interleukin-2 (IL2) receptor on T- and NK-cells. Studying clinical outcome and gene expression in multiple clinical cohorts, we found CD25 as a top-ranking predictor of poor clinical outcome in patients with B-ALL, mantle cell lymphoma and CLL.Our mechanistic experiments revealed that CD25 expressed on B-cells is monomeric. Unlike T- and NK-cells, CD25 expressed on activated or transformed B-cells does not form a heterotrimeric IL2 receptor with the IL2Rb and common g-chain. Instead of contributing to IL2-signaling, CD25 showed dynamic recruitment either to the B-cell receptor (BCR) or transforming oncogenes within lipid rafts. Conditional deletion of CD25 at earliest stages of B-cell development in mice resulted in profound B-cell defects. However, these defects were not replicated by IL2-deficiency and B-cell development in Il2-/- mice was normal.Inducible deletion of CD25 in genetic models for B-ALL and mantle cell lymphoma revealed an essential function of CD25 in maintaining homeostasis of signaling strength. In the absence of CD25, B-ALL and mantle cell lymphoma cells showed autonomous Ca 2+ oscillations, constitutive activation of Src-family kinases, BTK and ERK as well as the AMPK energy-stress sensor. CD25-deficient B-ALL and mantle cell lymphoma cells initially showed increased proliferation but rapidly died from exhaustion. Consistent with massive accumulation of p53, CD25-deficient B-ALL cells failed to form colonies and to initiate leukemia in transplant-recipient mice.Our mechanistic studies focused on the short (13 residues) cytoplasmic tale of CD25, which features a prominent serine/threonine kinase substrate motif. In vitro kinase assays for 62 candidate serine/threonine kinases revealed PKCd as top-ranking kinase and the serine residue 268 in the tail of CD25 as its principal substrate. The proximity-based labeling assays (BioID) revealed that the CD25 tail recruits PKCd and its adapter RACK1, which scaffold the inhibitory phosphatases SHIP1 and SHP1 for site-specific activation in lipid rafts (see Schematic). Consistent with these results, phospho-proteomic studies revealed that inducible deletion of CD25 resulted in acute inactivation of SHIP1 and SHP1 as well as the inhibitory RACK1 scaffold. Multiscale molecular dynamics (MD) simulations revealed that PKCd-mediated phosphorylation of the CD25-tail (S268) tightened interactions with RACK1 and SHIP1 and SHP1 phosphatases, whereas these interactions were destabilized when the CD25-tail was mutated (S268A).Conclusions: Mechanisms of site-specific recruitment of kinases for signal amplification have been extensively studied. However, structural elements that direct inhibitory phosphatases to sites of maximal signaling activity for feedback control remained largely unknown. Here we discovered a PKCd substrate motif in the cytoplasmic tail of CD25 (S268) as a central element to recruit inhibitory phosphatases, scaffolded by RACK1, to curb excessive signaling activity and energy expenditure. High expression levels of CD25 enable higher baseline activity of oncogenic signaling and are associated with poor patient-outcome. Our preclinical studies based on CD25-antibody-drug-conjugates (ADC) revealed that CD25 as a novel target in refractory B-ALL and mantle cell lymphoma. [Display omitted] DisclosuresWeinstock: SecuraBio: Consultancy; ASELL: Consultancy; Bantam: Consultancy; Abcuro: Research Funding; Verastem: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; AstraZeneca: Consultancy; Travera: Other: Founder/Equity; Ajax: Other: Founder/Equity.

Targeting enhancer reprogramming to mitigate MEK inhibitor resistance in preclinical models of advanced ovarian cancer.

Ovarian cancer is characterized by aberrant activation of the mitogen-activated protein kinase (MAPK), highlighting the importance of targeting the MAPK pathway as an attractive therapeutic strategy. However, the clinical efficacy of MEK inhibitors is limited by intrinsic or acquired drug resistance. Here, we established patient-derived ovarian cancer models resistant to MEK inhibitors and demonstrated that resistance to the clinically approved MEK inhibitor trametinib was associated with enhancer reprogramming. We also showed that enhancer decommissioning induced the downregulation of negative regulators of the MAPK pathway, leading to constitutive ERK activation and acquired resistance to trametinib. Epigenetic compound screening uncovered that HDAC inhibitors could alter the enhancer reprogramming and upregulate the expression of MAPK negative regulators, resulting in sustained MAPK inhibition and reversal of trametinib resistance. Consequently, a combination of HDAC inhibitor and trametinib demonstrated a synergistic antitumor effect in vitro and in vivo, including patient-derived xenograft mouse models. These findings demonstrated that enhancer reprogramming of the MAPK regulatory pathway might serve as a potential mechanism underlying MAPK inhibitor resistance and concurrent targeting of epigenetic pathways and MAPK signaling might provide an effective treatment strategy for advanced ovarian cancer.

Immunomodulatory Properties of BRAF and MEK Inhibitors Used for Melanoma Therapy-Paradoxical ERK Activation and Beyond.

The advent of mitogen-activated protein kinase (MAPK) inhibitors that directly inhibit tumor growth and of immune checkpoint inhibitors (ICI) that boost effector T cell responses have strongly improved the treatment of metastatic melanoma. In about half of all melanoma patients, tumor growth is driven by gain-of-function mutations of BRAF (v-rat fibrosarcoma (Raf) murine sarcoma viral oncogene homolog B), which results in constitutive ERK activation. Patients with a BRAF mutation are regularly treated with a combination of BRAF and MEK (MAPK/ERK kinase) inhibitors. Next to the antiproliferative effects of BRAF/MEKi, accumulating preclinical evidence suggests that BRAF/MEKi exert immunomodulatory functions such as paradoxical ERK activation as well as additional effects in non-tumor cells. In this review, we present the current knowledge on the immunomodulatory functions of BRAF/MEKi as well as the non-intended effects of ICI and discuss the potential synergistic effects of ICI and MAPK inhibitors in melanoma treatment.

Comprehensive Investigation of White Blood Cell and Gene Expression Profiles As Risk Factors for Multiple Myeloma in African Americans

Comprehensive Investigation of White Blood Cell and Gene Expression Profiles As Risk Factors for Multiple Myeloma in African Americans

Overactive IGF1/Insulin Receptors and NRASQ61R Mutation Drive Mechanisms of Resistance to Pazopanib and Define Rational Combination Strategies to Treat Synovial Sarcoma.

Pazopanib is approved for treatment of advanced soft tissue sarcomas, but primary and secondary drug resistance limits its clinical utility. We investigated the molecular mechanisms mediating pazopanib resistance in human synovial sarcoma (SS) models. We found reduced cell sensitivity to pazopanib associated with inefficient inhibition of the two critical signaling nodes, AKT and ERKs, despite strong inhibition of the main drug target, PDGFRα. In the CME-1 cell line, overactivation of IGF1 and Insulin receptors (IGF1R/InsR) sustained AKT activation and pazopanib resistance, which was overcome by a combination treatment with the double IGF1R/InsR inhibitor BMS754807. In the highly pazopanib resistant MoJo cell line, NRASQ61R mutation sustained constitutive ERK activation. Transfection of the NRAS mutant in the pazopanib sensitive SYO-1 cell line increased the drug IC50. MoJo cells treatment with pazopanib in combination with the MEK inhibitor trametinib restored ERK inhibition, synergistically inhibited cell growth, and induced apoptosis. The combination significantly enhanced the antitumor efficacy against MoJo orthotopic xenograft abrogating growth in 38% of mice. These findings identified two different mechanisms of intrinsic pazopanib resistance in SS cells, supporting molecular/immunohistochemical profiling of tumor specimens as a valuable approach to selecting patients who may benefit from rational drug combinations.

Co-Expression of CD56 and Production of Transforming Growth Factor Beta By Foxp3 Regulatory T Cells from Langerhans Cell Histiocytosis Lesions

Introduction:Langerhans cell histiocytosis (LCH) is a rare disease involving inflammatory lesions that can occur in essentially any organ of the body. LCH lesions are defined by the presence of CD1a+/CD207+ myeloid lineage cells (LCH cells), which often have mutations within the RAS/RAF/MEK/ERK pathway and constitutive activation of ERK. A range of other immune cells are also present within lesions including an enrichment of Foxp3 regulatory T cells (Tregs).The immune suppressive cytokine transforming growth factor beta (TGF-β), which is commonly produced by Foxp3 Tregs has also been detected within lesions and blood from patients with active LCH disease. Groups have suggested that LCH cells are a source of TGF-β and that TGF-β is one of the drivers of the LCH cell phenotype. Given the enrichment of Foxp3 Tregs and the presence of TGF-β within LCH lesions it is conceivable that Foxp3 Tregs too are a source of TGF-β production.Historically the identification of Foxp3 Tregs has not allowed for functional studies to be conducted because staining for the Foxp3 transcription factor requires cell permeabilization. A newer surrogate gating strategy to detect CD3+CD4+CD25+CD127low lymphocytes allows for downstream functional assays on the Foxp3 Treg population. Our study aimed to better define the role of Tregs in LCH pathogenesis using this surrogate staining method.Results:A surrogate Treg identification method was employed to gate on CD3+CD4+CD25+CD127low lymphocytes in the blood from healthy donors and patients with LCH and in lesions from patients with LCH. Using this gating strategy we identified similarly to previous studies that the proportion of lesional Tregs (median = 12.85 %, IQR = 7.85-26.72 %, n = 6) was significantly (p < 0.0001) increased in the total T cell population when compared to those in the blood from healthy donors (median = 1.02 %, IQR = 0.84-1.20 %, n = 19). The proportion of Tregs in blood from patients with active LCH (median = 1.73 %, IQR = 1.58-3.80 %, n = 8) was also significantly (p = 0.232) increased when compared to those in the blood from healthy donors. We confirmed Foxp3 expression in Tregs in lesions from three independent LCH donors by staining for the transcription factor, and we confirmed that other lesional CD4+ T cells were mostly negative for Foxp3.Due to our interest in unconventional T cells we analysed specimens for CD56 expression in conjunction with investigating Tregs and we unexpectedly found CD56 expression on a considerable proportion of the Treg population in LCH lesions (median = 36.48 %, IQR = 24.94-55.14 %, n = 6). The proportion of Tregs that displayed CD56 expression on their cell surface was significantly higher in the lesions from patients with LCH compared to the blood from healthy donors (median = 2.29 %, IQR = 0.61-4.10 %, n = 15, p = 0.0092) and patients with active LCH (median = 0.57 %, IQR = 0.11-0.85 %, n = 8, p < 0.0001). Additionally, we found that the proportion of CD56+ Tregs in total T cells from LCH lesions was directly correlated to the proportion of total Tregs in overall lesional T cells (r = 1, p = 0.0028). Importantly, we confirmed Foxp3 expression by both the CD56+ and CD56- Treg populations in lesions from three independent LCH donors.We purified and then stimulated Tregs from healthy donors, and CD56+ and CD56- Treg populations from lesions from patients with LCH. Using supernatants from this assay we found that Tregs from lesions from patients with LCH produced active TGF-β when challenged with PMA and ionomycin for 16 hours (CD56+ Tregs n = 3, CD56- Tregs n = 3).Conclusion:We have confirmed that both CD56+ and CD56- Treg populations from the lesions from patients with LCH are able to produce TGF-β. Given their cytokine potential, the functional status of Tregs in LCH lesions is therefore most likely to be suppressive in nature. This means that Tregs are possibly responsible for a component of the well documented expression of TGF-β within lesions. DisclosuresNo relevant conflicts of interest to declare.

Comparative Characterization of Osteoclasts Derived From Murine Bone Marrow Macrophages and RAW 264.7 Cells Using Quantitative Proteomics.

ABSTRACTOsteoclasts are bone‐resorbing cells differentiated from macrophage/monocyte precursors in response to macrophage colony‐stimulating factor (M‐CSF) and receptor activator of NF‐κB ligand (RANKL). In vitro models are principally based on primary bone marrow macrophages (BMMs), but RAW 264.7 cells are frequently used because they are widely available, easy to culture, and more amenable to genetic manipulation than primary cells. Increasing evidence, however, has shown that the vastly different origins of these two cell types may have important effects on cell behavior. In particular, M‐CSF is a prerequisite for the differentiation of BMMs, by promoting survival and proliferation and priming the cells for RANKL induction. RAW 264.7 cells readily form osteoclasts in the presence of RANKL, but M‐CSF is not required. Based on these key differences, we sought to understand their functional implications and how it might affect osteoclast differentiation and related signaling pathways. Using a robust and high‐throughput proteomics strategy, we quantified the global protein changes in osteoclasts derived from BMMs and RAW 264.7 cells at 1, 3, and 5 days of differentiation. Data are available via ProteomeXchange with the identifier PXD009610. Correlation analysis of the proteomes demonstrated low concordance between the two cell types (R2 ≈ 0.13). Bioinformatics analysis indicate that RANKL‐dependent signaling was intact in RAW 264.7 cells, but biological processes known to be dependent on M‐CSF were significantly different, including cell cycle control, cytoskeletal organization, and apoptosis. RAW 264.7 cells exhibited constitutive activation of Erk and Akt that was dependent on the activity of Abelson tyrosine kinase, and the timing of Erk and Akt activation was significantly different between BMMs and RAW 264.7 cells. Our findings provide the first evidence for major discrepancies between BMMs and RAW 264.7 cells, indicating that careful consideration is needed when using the RAW 264.7 cell line for studying M‐CSF‐dependent signaling and functions. © 2018 American Society for Bone and Mineral Research. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Concurrent activation of β2-adrenergic receptor and blockage of GPR55 disrupts pro-oncogenic signaling in glioma cells

Activation of β2-adrenergic receptor (β2AR) and deorphanized GPR55 has been shown to modulate cancer growth in diverse tumor types in vitro and in xenograft models in vivo. (R,R′)-4′-methoxy-1-naphthylfenoterol [(R,R′)-MNF] is a bivalent compound that agonizes β2AR but inhibits GPR55-mediated pro-oncogenic responses. Here, we investigated the molecular mechanisms underlying the anti-tumorigenic effects of concurrent β2AR activation and GPR55 blockade in C6 glioma cells using (R,R′)-MNF as a marker ligand. Our data show that (R,R′)-MNF elicited G1-phase cell cycle arrest and apoptosis, reduced serum-inducible cell motility, promoted the phosphorylation of PKA target proteins, and inhibited constitutive activation of ERK and AKT in the low nanomolar range, whereas high nanomolar levels of (R,R′)-MNF were required to block GPR55-mediated cell motility. siRNA knockdown and pharmacological inhibition of β2AR activity were accompanied by significant upregulation of AKT and ERK phosphorylation, and selective alteration in (R,R′)-MNF responsiveness. The effects of agonist stimulation of GPR55 on various readouts, including cell motility assays, were suppressed by (R,R′)-MNF. Lastly, a significant increase in phosphorylation-mediated inactivation of β-catenin occurred with (R,R′)-MNF, and we provided new evidence of (R,R′)-MNF-mediated inhibition of oncogenic β-catenin signaling in a C6 xenograft tumor model. Thus, simultaneous activation of β2AR and blockade of GPR55 may represent a novel therapeutic approach to combat the progression of glioblastoma cancer.

Prominent Oncogenic Roles of EVI1 in Breast Carcinoma.

Overexpression of the EVI1 oncogene is associated typically with aggressive myeloid leukemia, but is also detectable in breast carcinoma where its contributions are unexplored. Analyzing a tissue microarray of 608 breast carcinoma patient specimens, we documented EVI1 overexpression in both estrogen receptor-positive (ER+) and estrogen receptor-negative (ER-) breast carcinomas. Here, we report prognostic relevance of EVI1 overexpression in triple-negative breast carcinoma but not in the HER2-positive breast carcinoma subset. In human breast cancer cells, EVI1 silencing reduced proliferation, apoptosis resistance, and tumorigenicity, effects rescued by estrogen supplementation in ER+ breast carcinoma cells. Estrogen addition restored ERK phosphorylation in EVI1-silenced cells, suggesting that EVI1 and estradiol signaling merge in MAPK activation. Conversely, EVI1 silencing had no effect on constitutive ERK activity in HER2+ breast carcinoma cells. Microarray analyses revealed G-protein-coupled receptor (GPR) signaling as a prominent EVI1 effector mechanism in breast carcinoma. Among others, the GPR54-ligand KISS1 was identified as a direct transcriptional target of EVI1, which together with other EVI1-dependent cell motility factors such as RHOJ regulated breast carcinoma cell migration. Overall, our results establish the oncogenic contributions of EVI1 in ER- and HER2-negative subsets of breast cancer. Cancer Res; 77(8); 2148-60. ©2017 AACR.

ERK and p38 MAPK Activities Determine Sensitivity to PI3K/mTOR Inhibition via Regulation of MYC and YAP.

Aberrant activation of the PI3K/mTOR pathway is a common feature of many cancers and an attractive target for therapy, but resistance inevitably evolves as is the case for any cancer cell-targeted therapy. In animal tumor models, chronic inhibition of PI3K/mTOR initially inhibits tumor growth, but over time, tumor cells escape inhibition. In this study, we identified a context-dependent mechanism of escape whereby tumor cells upregulated the proto-oncogene transcriptional regulators c-MYC and YAP1. This mechanism was dependent on both constitutive ERK activity as well as inhibition of the stress kinase p38. Inhibition of p38 relieved proliferation arrest and allowed upregulation of MYC and YAP through stabilization of CREB. These data provide new insights into cellular signaling mechanisms that influence resistance to PI3K/mTOR inhibitors. Furthermore, they suggest that therapies that inactivate YAP or MYC or augment p38 activity could enhance the efficacy of PI3K/mTOR inhibitors. Cancer Res; 76(24); 7168-80. ©2016 AACR.

Measurement of Phosphorylated ERK As a Prognostic and Predictive Marker for MEK Inhibition in Pediatric B-Lymphoblastic Leukemia: A Pilot Study

Abstract Background: Outcomes for patients with relapsed B- acute lymphoblastic leukemia (B-ALL) remain suboptimal. We and others have identified the Mitogen activated protein kinase/ Extracellular signal regulated kinase (MAPK/ERK) pathway as a primary driver of drug resistance in pediatric B-ALL. Constitutive activation of ERK has been found to be an independent predictor of complete response and is associated with other poor prognostic factors in adults with B-ALL. Mutations that affect activation of the Ras/Raf/MEK/ERK pathway have been found in up to 40% of children with newly diagnosed and relapsed ALL and these mutations correlate with early relapse and poor overall survival. However, we have demonstrated MAPK activation in the absence of Ras pathway mutations, suggesting alternative mechanisms of activation. Hypothesis: We hypothesize that activation of the MAPK pathway drives drug resistance in pediatric B-ALL. Activation of this pathway at diagnosis may portend a high likelihood of relapse and inhibition of the MAPK pathway may restore chemosensitivity to conventional agents. Methods: For our initial pilot study, a total of 24 patient samples from initial diagnosis obtained from Children's Oncology Group cell bank and from NYU, were thawed and placed either in serum-free media or media containing Trametinib (MEK1/2 inhibitor) at 1µM for 4 hours. Samples were then stimulated with Phorbol 12-myristate 13-acetate (PMA), a specific activator of Protein Kinase C, which activates MEK via Ras. Samples were then fixed with 4% paraformaldehyde, permeabilized with 95% methanol, and stained for Caspase 3, CD10, phosphorylated ERK (p-ERK), and p-AKT. All samples were simultaneously processed on the BD LSRII HTS and analyzed on FlowJo software (Treestar, V.10). Viable leukemic blasts were identified as CD10 positive and Caspase 3 negative cells, which were gated on for further analysis. Median fluorescence intensity (MFI) for phosphorylated proteins was measured, expressed as a ratio to isotype controls and compared between samples. MAPK activation was defined by maximal p-ERK levels upon stimulation with PMA. Inhibition was quantified as percentage decrease in maximal p-ERK levels (upon PMA stimulation) after treatment with Trametinib. Targeted Sanger sequencing of commonly mutated exons of Ras pathway genes (NRAS, KRAS, FLT3 and PTPN11) was performed on 14 samples based on availability of DNA. Results: Of our 24 samples, 4 samples were excluded from analysis due to poor viability (&lt;40% live cells), giving an assay success rate of 83.3%. We were able to successfully measure p-ERK in all 20 viable patient samples and also measure levels of baseline p-AKT in 16 samples. A wide range of p-ERK levels and MAPK activation was observed within patient samples (median activation ratio 12.3, range 4.4-44.7) and 7 samples showed markedly higher levels of activation with ratios between 21.9 - 44.78. Levels of inhibition by Trametinib ranged from 0-82.2%, with a median of 43%. Greater degree of MAPK activation was found to correlate strongly with higher levels of inhibition by Trametinib (overall correlation coefficient 0.72). In 2 samples, the levels of inhibition by Trametinib seemed lower than anticipated for levels of MAPK activation, but both of these samples showed higher than average levels of p-AKT (p-AKT to isotype ratio of 12.3-12.9, median of 2.4 in rest of the samples), suggesting a possible alternative mechanism of activation via the PI3K/AKT signaling pathway. DNA was obtained for Sanger sequencing in 14 of the 20 viable patient samples. Three of the 14 samples had point mutations in Ras pathway genes of which two had mutations in exon 2 of NRAS while one had a mutation in exon 14 of FLT3. Two of the seven patient samples with the highest MAPK activation had Ras mutations, which is consistent with our prior data showing that additional mechanisms of pathway activation are operative. Conclusion: We have successfully established a phospho-flow protocol that enables the measurement of MAPK activation in real time in patient samples. Based on our pilot data, we also conclude that MAPK activation in itself serves as a better predictive marker for susceptibility to MAPK inhibition, as opposed to Ras pathway mutation status alone. Ongoing investigation will allow correlation with outcome and the assay could serve in the future to identify those patients who might benefit from MEK inhibition or other nodes in the pathway. Disclosures Loh: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding.

The plasma membrane Ca2+ pump PMCA4b inhibits the migratory and metastatic activity of BRAF mutant melanoma cells.

Oncogenic mutations of BRAF lead to constitutive ERK activity that supports melanoma cell growth and survival. While Ca2+ signaling is a well-known regulator of tumor progression, the crosstalk between Ca2+ signaling and the Ras-BRAF-MEK-ERK pathway is much less explored. Here we show that in BRAF mutant melanoma cells the abundance of the plasma membrane Ca2+ ATPase isoform 4b (PMCA4b, ATP2B4) is low at baseline but markedly elevated by treatment with the mutant BRAF specific inhibitor vemurafenib. In line with these findings gene expression microarray data also shows decreased PMCA4b expression in cutaneous melanoma when compared to benign nevi. The MEK inhibitor selumetinib-similarly to that of the BRAF-specific inhibitor-also increases PMCA4b levels in both BRAF and NRAS mutant melanoma cells suggesting that the MAPK pathway is involved in the regulation of PMCA4b expression. The increased abundance of PMCA4b in the plasma membrane enhances [Ca2+ ]i clearance from cells after Ca2+ entry. Moreover we show that both vemurafenib treatment and PMCA4b overexpression induce marked inhibition of migration of BRAF mutant melanoma cells. Importantly, reduced migration of PMCA4b expressing BRAF mutant cells is associated with a marked decrease in their metastatic potential in vivo. Taken together, our data reveal an important crosstalk between Ca2+ signaling and the MAPK pathway through the regulation of PMCA4b expression and suggest that PMCA4b is a previously unrecognized metastasis suppressor.