Case Report: Opposite Effects of BRAF Inhibition on Closely Related Clonal Myeloid Disorders

Langerhans cell histiocytosis (LCH) commonly co-occurs with additional myeloid malignancies. The introduction of targeted therapies, blocking “driver” mutations (e.g., BRAF V600E), enabled long-term remission in patients with LCH. The effect of BRAF inhibition on the course and the prognosis of co-existing clonal hematopoiesis is poorly understood. We report on a 61-year-old patient with systemic BRAF V600E positive LCH and concomitant BRAF wild-type (wt) clonal cytopenia of unknown significance (CCUS) with unfavorable somatic mutations including loss of function (LOF) of NF1. While manifestations of LCH improved after blocking BRAF by dabrafenib treatment, the BRAF wt CCUS progressed to acute myeloid leukemia (AML). The patient eventually underwent successful allogeneic hematopoietic stem cell transplantation (HSCT). We performed an in-depth analyzes of the clonal relationship of CCUS and the tissue affected by LCH by using next-generation sequencing (NGS). The findings suggest activation of the mitogen-activated protein (MAP) kinase pathway in the CCUS clone due to the presence of the RAS deregulating NF1 mutations and wt BRAF, which is reportedly associated with paradoxical activation of CRAF and hence MEK. Patients with LCH should be carefully screened for potential additional clonal hematological diseases. NGS can help predict outcome of the latter in case of BRAF inhibition. Blocking the MAP kinase pathway further downstream (e.g., by using MEK inhibitors) or allogeneic HSCT may be options for patients at risk.

Coordinated Regulation of Protoperithecium Development by MAP Kinases MAK-1 and MAK-2 in Neurospora crassa

Mitogen-activated protein (MAP) kinase pathways function as signaling hubs that are integral for many essential cellular processes, including sexual development. The molecular mechanisms and cross-talk between PR and CWI MAP kinase pathways have been extensively studied during asexual development. However, if these can be extended to sexual development remains elusive. By analyzing genome-wide transcriptional responses to deletion of each of two MAP kinase coding genes mak-2 (PR-MAP kinase pathway) and mak-1 (CWI-MAP kinase pathway) in Neurospora crassa during protoperithecium formation, 430 genes co-regulated by the MAK-1 and MAK-2 proteins were found, functionally enriched at integral components of membrane and oxidoreductase. These genes include 13 functionally known genes participating in sexual development (app, poi-2, stk-17, fsd-1, vsd-8, and NCU03863) and melanin synthesis (per-1, pkh-1, pkh-2, mld-1, scy-1, trn-2, and trn-1), as well as a set of functionally unknown genes. Phenotypic analysis of deletion mutants for the functionally unknown genes revealed that 12 genes were essential for female fertility. Among them, single-gene deletion mutants for NCU07743 (named as pfd-1), NCU02250 (oli), and NCU05948 (named as pfd-2) displayed similar protoperithecium development defects as the Δmak-1 and Δmak-2 mutants, failing to form protoperithecium. Western blotting analysis showed that both phosphorylated and total MAK-1 proteins were virtually abolished in the Δnrc-1, Δmek-2, and Δmak-2 mutants, suggesting that the posttranscriptional regulation of MAK-1 is dependent on the PR-MAP kinase pathway during the protoperithecium development. Taken together, this study revealed the regulatory roles and cross-talk between PR and CWI-MAP kinase pathways during protoperithecium development.

Case Report: Evolution of a Severe Vascular Refractory Form of ECD Requiring Liver Transplantation Correlated With the Change in the Monocyte Subset Analysis

Erdheim–Chester disease is a rare histiocytosis characterized by iconic features associated with compatible histology. Most patients have somatic mutations in the MAP-kinase pathway gene, and the mutations occur in CD14+ monocytes. Differentiation of the myeloid lineage plays a central role in the pathogenesis of histiocytosis. Monocytes are myeloid-derived white blood cells, divided into three subsets, but only the CD14++CD16− “classical monocyte” can differentiate into dendritic cells and tissue macrophages. Since most mutations occur in CD14+ cells and since ECD patients have a particular monocytic phenotype resembling CMML, we studied the correlation between disease activity and monocytic subset distribution during the course of a severe vascular form of ECD requiring liver transplantation. During early follow-up, increased CD14++CD16− “classical monocyte” associated with decreased CD14lowCD16++ “non-classical monocyte” correlated with disease activity. Further studies are needed to confirm the use of monocyte as a marker of disease activity in patients with ECD.

Real World Outcomes in Patients with Advanced Melanoma Treated in Alberta, Canada: A Time-Era Based Analysis

Immune checkpoint and MAP kinase pathway inhibitors can significantly improve long-term survival for patients with melanoma. There is limited real-world data of these regimens’ effectiveness. We retrospectively analyzed 402 patients with unresectable and metastatic melanoma between August 2013 and July 2020 treated with immune checkpoint inhibitors and MAP kinase pathway targeted therapy in Alberta, Canada. Overall survival (OS) was compared using Kaplan–Meier and Cox regression analyses. Subgroup survival outcomes were analyzed by first-line treatment regime and BRAF mutation status. Three treatment eras were defined based on drug access: prior to August 2013, August 2013 to November 2016, and November 2016 to July 2020. Across each era, there were improvements in median OS: 11.7 months, 15.9 months, and 33.6 months, respectively. Patients with BRAF mutant melanoma had improved median OS when they were treated with immunotherapy in the first line as opposed to targeted therapy (median OS not reached for immunotherapy versus 17.4 months with targeted treatment). Patients with BRAF wild-type melanomas had improved survival with ipilimumab and nivolumab versus those treated with a single-agent PD-1 inhibitor (median OS not reached and 21.2 months). Our real-world analysis confirms significant survival improvements with each subsequent introduction of novel therapies for advanced melanoma.

Activation of the CaMKII-Sarm1-ASK1 MAP kinase pathway protects against axon degeneration caused by loss of mitochondria

Mitochondrial defects are tightly linked to axon degeneration, yet the underlying cellular mechanisms remain poorly understood. In C. elegans, PVQ axons that lack mitochondria degenerate spontaneously with age. Using an unbiased genetic screen, we found that cell-specific activation of CaMKII/UNC-43 suppresses axon degeneration due to loss of mitochondria. Unexpectedly, CaMKII/UNC-43 protects against degeneration through the conserved Sarm1/TIR-1-ASK1/NSY-1 MAPK pathway. In addition, we show that disrupting a trafficking complex composed of calsyntenin/CASY-1, Mint/LIN-10, and kinesin suppresses axon degeneration. Further analysis indicates that disruption of this trafficking complex activates the CaMKII-Sarm1-MAPK pathway through L-type voltage-gated calcium channels. Our findings identify CaMKII as a pivot point between mitochondrial defects and axon degeneration, describe how it is regulated in this context, and uncover a surprising neuroprotective role for the Sarm1-ASK1 pathway.

A Rare Case of Serrated Polyposis Syndrome with the MSH6 and SMARCA4 variants

Serrated polyposis syndrome (SPS) is the most common form of polyposis syndrome and has been shown to increase the risk of colorectal cancer (CRC). The genetic pathway of CRC in SPS is different from the classic adenomatous polyposis coli (APC) pathway, which accounts for 70–80% of cases of CRC. Most commonly, SPS mutations include BRAF and KRAS, with activation of the RAS-RAF-MAP kinase pathway involved in the pathogenesis of serrated lesions. We present a rare case of SPS in a 32-year-old woman with MSH6 and SMARCA4 variants, which have not previously been reported in the literature.

Turnover Regulation of the Rho GTPase Cdc42 by Heat Shock Protein Chaperones and the MAPK Pathway Scaffold Bem4

All cells maintain an axis of polarity that directs the orientation of growth. Cell polarity can be reorganized during development and in response to extrinsic cues to produce new cell types. Rho GTPases are central regulators of cell polarity and signal-dependent cell differentiation. We show here that one of the best understood Rho GTPases, the highly conserved yeast Cdc42p, is turned over by members of the Heat Shock family of Proteins (HSPs). The Hsp40p chaperone, Ydj1p, was required for turnover of Cdc42p by the NEDD4 E3 ubiquitin ligase, Rsp5p, in the proteosome. Cdc42p turnover was regulated by HSPs at high temperatures, and in aging cells where the protein formed aggregates, implicating HSPs in Rho GTPase quality control. We also show that Cdc42pQ61L, which mimics the active (GTP-bound) conformation of the protein, was turned over at elevated levels by Ydj1p and Rsp5p. A turnover-defective version of Cdc42pQ61L led to multibudding phenotypes, implicating Cdc42 turnover in singularity in cell polarization. Cdc42p turnover also impacted MAP kinase pathway specificity. A pathway-specific scaffold, Bem4p, stabilized Cdc42p levels, which biased Cdc42p function in one MAPK pathway over another. Turnover regulation of Rho GTPases by HSPs and scaffolds provides new dimensions to the regulation of cell polarity and signal-dependent morphogenesis.

NGS-Based Analysis of Atypical Deep Penetrating Nevi

Deep penetrating nevi (DPNs) are rare melanocytic neoplasms consisting of pigmented spindled or epithelioid melanocytes with a distinctive wedge-shaped configuration showing activation of the WNT pathway, with unusual cyto-architectural features. It is unclear whether they show a distinct genomic profile associated with a diverse metastatic potential. We describe herein a cohort of 21 atypical DPNs analyzed by next-generation sequencing using the Ion AmpliSeq™ Comprehensive Cancer Panel. We found that β-catenin exon 3 was mutated in 95% and MAP kinase pathway genes in 71% of the cases. Less frequent mutations were observed in HRAS (19%) and MAP2K1 (24%). Isocitrate dehydrogenases 1 (IDH1) mutations, including R132C, V178I, and S278L, were identified in 38% of cases and co-existed with BRAF/HRAS mutations. The only case with progressive nodal disease carried alterations in the β-catenin pathway and mutations in IDH1 and NRAS (codon 61). By a comprehensive mutation analysis, we found low genetic heterogeneity and a lack of significant associations between specific gene mutations and histopathological features, despite atypical features. Whether the acquisition of an NRAS or IDH1 mutation in an atypical DPN may represent a molecular evolution implying a pathway to melanoma progression should be confirmed in a larger series.

Loperamide-induced Constipation activates Inflammatory Signaling Pathways in the Transverse Colon of SD rats via Complement C3 and its receptors

Background: Complement component 3 (C3) receptors play an important role as an inflammatory mediator in the innate immune system, although their mechanism was not well studied during constipation. Objective: The aim of this study is to investigate the regulatory role of C3 and its receptors' downstream signaling during constipation. Methods: Alterations in the C3, C3a receptor (C3aR), and C3b receptor (C3bR) expressions, PI3K/AKT pathway, RhoA/MLC pathway, MAP kinase pathway, and inflammatory cytokine expressions were measured in the transverse colon of loperamide (Lop) treated SD rats. Results: Lop treatment successfully induced constipation phenotypes, including decreased stool parameters and histological structure alterations. The expression levels of C3 were significantly increased, whereas expressions of C3aR and C3bR were observed to decrease during Lop-induced constipation. Moreover, significant upregulation was observed in the phosphorylation levels of PI3K, AKT, and GSK3 in transverse colons of Lop treated SD rats. The expression of RhoA and phosphorylation of MLC were also enhanced in the Lop treated group. Furthermore, a similar pattern was detected in the MAP kinase pathway and inflammatory cytokine expressions. Subsequent to Lop treatment, the phosphorylation of ERK and p38, as well as the mRNA levels of NF-B, TNF-, IL-6 and IL-1 were remarkably increased in the transverse colon. Conclusion: These results indicate that Lop-induced constipation is tightly linked to the downregulation of C3aR and C3bR expressions and upregulation of the C3, C3Rs downstream signaling pathway, including PI3K/AKT, RhoA/MLC, and MAP kinase pathways as well as inflammatory cytokine expressions in the transverse colon of SD rats.