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Invasive breast carcinoma is a combination of heterogeneous diseases with distinct molecular and clinical features. Some subsets of breast cancer present major clinical challenges, including triple-negative, metastatic/recurrent disease and rare breast histologies. Previously, we developed a unique resource of 37 hard-to-treat breast cancer patient-derived xenografts (PDX). This set included mainly triple negative breast cancer (TNBC) patients that presented poor response to neoadjuvant chemotherapies (Savage et al. 2020 - PMID: 32546838). PDXs accurately reproduce the molecular heterogeneity of the primary tumors and show that multi-drug chemoresistance was retained upon xenotransplantation. Here, we present the characterization of PDX 3-dimensional cultures organoids (8) and PDX derived epithelial cell lines (11). Using single-cell RNAseq we showed that an organoid cultured for several passages (P8) maintained the heterogeneity of the matched PDX. Although in different proportions, all cancer cell populations found in the PDX were retained in matched organoids supporting that organoids are suitable models that recapitulates the tumor heterogeneity and are therefore a suitable model for drug screening. Among our new PDXs models (30), we have developed four PDXs from rare metaplastic breast cancers (MpBC), an aggressive subtype of breast cancer that present the poorest response to standard of care chemotherapy. We also developed one male breast cancer PDX with matched organoid. Omic analysis on our pre-clinical models and patient primary tumor and metastasis will inform development of therapeutic opportunities. This molecular information will guide selection of compounds that will be validated using our high throughput organoid drug screening pipeline. This will allow rapid screens of thousands of approved drugs, enhancing drug repurposing with potential for rapid clinical translation. The combined use of 3D tumor organoids and PDXs, is an important opportunity poised to transform identification of new therapeutic options for hard-to-treat lethal breast cancers. Citation Format: Hellen Kuasne, Anne-Marie fortier, Sandrine Busque, Simon Mathien, Paul Savage, Constanza Martinez Ramirez, Anie Monast, Margarita Soleinova, Atilla Omeroglu, Jamil Asselah, Nathaniel Bouganim, Sarkis Meterissian, Mark Basik, Morag Park. Development and molecular characterization of hard-to-treat breast cancer pre-clinical models to enhance precision medicine [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-09.

Mitogen-activated protein kinase (MAPK) cascades are evolutionarily conserved signaling pathways that play essential roles in transducing extracellular environmental signals into diverse cellular responses to maintain homeostasis. These pathways are classically organized into an architecture of three sequentially acting protein kinases: a MAPK kinase kinase that phosphorylates and activates a MAPK kinase, which in turn phosphorylates and activates the effector MAPK. The activity of MAPKs is tightly regulated by phosphorylation of their activation loop, which can be modulated by positive and negative feedback mechanisms to control the amplitude and duration of the signal. The signaling outcomes of MAPK pathways are further regulated by interactions of MAPKs with scaffolding and regulatory proteins. Accumulating evidence indicates that, in addition to these mechanisms, MAPK signaling is commonly regulated by ubiquitin-proteasome system (UPS)-mediated control of the stability and abundance of MAPK pathway components. Notably, the biologic activity of some MAPKs appears to be regulated mainly at the level of protein turnover. Recent studies have started to explore the potential of targeted protein degradation as a powerful strategy to investigate the biologic functions of individual MAPK pathway components and as a new therapeutic approach to overcome resistance to current small-molecule kinase inhibitors. Here, we comprehensively review the mechanisms, physiologic importance, and pharmacological potential of UPS-mediated protein degradation in the control of MAPK signaling. Significance Statement Accumulating evidence highlights the importance of targeted protein degradation by the ubiquitin-proteasome system in regulating and fine-tuning the signaling output of mitogen-activated protein kinase (MAPK) pathways. Manipulating protein levels of MAPK cascade components may provide a novel approach for the development of selective pharmacological tools and therapeutics.

The physiological functions of the atypical mitogen-activated protein kinase extracellular signal-regulated kinase 3 (ERK3) remain poorly characterized. Previous analysis of mice with a targeted insertion of the lacZ reporter in the Mapk6 locus (Mapk6(lacZ)) showed that inactivation of ERK3 in Mapk6(lacZ) mice leads to perinatal lethality associated with intrauterine growth restriction, defective lung maturation, and neuromuscular anomalies. To further explore the role of ERK3 in physiology and disease, we generated novel mouse models expressing a catalytically inactive (Mapk6(KD)) or conditional (Mapk6(Δ)) allele of ERK3. Surprisingly, we found that mice devoid of ERK3 kinase activity or expression survive the perinatal period without any observable lung or neuromuscular phenotype. ERK3 mutant mice reached adulthood, were fertile, and showed no apparent health problem. However, analysis of growth curves revealed that ERK3 kinase activity is necessary for optimal postnatal growth. To gain insight into the genetic basis underlying the discrepancy in phenotypes of different Mapk6 mutant mouse models, we analyzed the regulation of genes flanking the Mapk6 locus by quantitative PCR. We found that the expression of several Mapk6 neighboring genes is deregulated in Mapk6(lacZ) mice but not in Mapk6(KD) or Mapk6(Δ) mutant mice. Our genetic analysis suggests that off-target effects of the targeting construct on local gene expression are responsible for the perinatal lethality phenotype of Mapk6(lacZ) mutant mice.

The physiological functions of the atypical MAP kinase ERK3 remain poorly characterized. Previous analysis of mice with a targeted insertion of the lacZ reporter in the Mapk6 locus (Mapk6lacZ) showed that inactivation of ERK3 in Mapk6lacZ mice leads to perinatal lethality associated with intrauterine growth restriction, defective lung maturation, and neuromuscular anomalies. To further explore the role of ERK3 in physiology and disease, we generated novel mouse models expressing a catalytically-inactive (Mapk6KD) or conditional (Mapk6Δ) allele of ERK3. Surprisingly, we found that mice devoid of ERK3 kinase activity or expression survive the perinatal period without any observable lung or neuromuscular phenotype. ERK3 mutant mice reached adulthood, were fertile and showed no apparent health problem. However, analysis of growth curves revealed that ERK3 kinase activity is ncessary for optimal post-natal growth. To gain insight into the genetic basis underlying the discrepancy in phenotypes of different Mapk6 mutant mouse models, we analyzed the regulation of genes flanking the Mapk6 locus by quantitative PCR. We found that expression of several Mapk6 neighboring genes is deregulated in Mapk6lacZ mice, but not in Mapk6KD or Mapk6Δ mutant mice. Our genetic analysis suggests that off-target effects of the targeting construct on local gene expression are likely to be responsible for the perinatal lethality phenotype of Mapk6lacZ mutant mice.

Review on MAPK6 (mitogen-activated protein kinase 6), with data on DNA, on the protein encoded, and where the gene is implicated.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK) whose regulatory mechanisms and biological functions remain superficially understood. Contrary to most protein kinases, ERK3 is a highly unstable protein that is subject to dynamic regulation by the ubiquitin-proteasome system. However, the effectors that control ERK3 ubiquitination and degradation are unknown. In this study, we carried out an unbiased functional loss-of-function screen of the human deubiquitinating enzyme (DUB) family and identified ubiquitin-specific protease 20 (USP20) as a novel ERK3 regulator. USP20 interacts with and deubiquitinates ERK3 both in vitro and in intact cells. The overexpression of USP20 results in the stabilization and accumulation of the ERK3 protein, whereas USP20 depletion reduces the levels of ERK3. We found that the expression levels of ERK3 correlate with those of USP20 in various cellular contexts. Importantly, we show that USP20 regulates actin cytoskeleton organization and cell migration in a manner dependent on ERK3 expression. Our results identify USP20 as a bona fide regulator of ERK3 stability and physiological functions.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family whose function is largely unknown. Given the central role of MAPKs in T cell development, we hypothesized that ERK3 may regulate thymocyte development. Here we have shown that ERK3 deficiency leads to a 50% reduction in CD4(+) CD8(+) (DP) thymocyte number. Analysis of hematopoietic chimeras revealed that the reduction in DP thymocytes is intrinsic to hematopoietic cells. We found that early thymic progenitors seed the Erk3(-/-) thymus and can properly differentiate and proliferate to generate DP thymocytes. However, ERK3 deficiency results in a decrease in the DP thymocyte half-life, associated with a higher level of apoptosis. As a consequence, ERK3-deficient DP thymocytes are impaired in their ability to make successful secondary T cell receptor alpha (TCRα) gene rearrangement. Introduction of an already rearranged TCR transgene restores thymic cell number. We further show that knock-in of a catalytically inactive allele of Erk3 fails to rescue the loss of DP thymocytes. Our results uncover a unique role for ERK3, dependent on its kinase activity, during T cell development and show that this atypical MAPK is essential to sustain DP survival during RAG-mediated rearrangements.

The classical mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 are activated upon stimulation of cells with a broad range of extracellular signals (including antigens) allowing cellular responses to occur. ERK3 is an atypical member of the MAPK family with highest homology to ERK1/2. Therefore, we evaluated the role of ERK3 in mature T cell response. Mouse resting T cells do not transcribe ERK3 but its expression is induced in both CD4+ and CD8+ T cells following T cell receptor (TCR)-induced T cell activation. This induction of ERK3 expression in T lymphocytes requires activation of the classical MAPK ERK1 and ERK2. Moreover, ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. We show that ERK3-deficient T cells have a decreased proliferation rate and are impaired in cytokine secretion following in vitro stimulation with low dose of anti-CD3 antibodies. Our findings identify the atypical MAPK ERK3 as a new and important regulator of TCR-induced T cell activation.