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Background KMT2A-rearranged (KMT2A-R) is an aggressive and chemo-refractory acute leukemia which mostly affects children. Transcriptomics-based characterization and chemical interrogation identified kinases as key drivers of survival and drug resistance in KMT2A-R leukemia. In contrast, the contribution and regulation of phosphatases is unknown. We explored the role of SET, the endogenous inhibitor of SER/THR phosphatase PP2A in KMT2A-R leukemia. Material and Methods The expression of SET was analysed in a large acute myeloid leukemia (AML)- RNA-seq dataset and in primary KMT2A-R samples and aged matched-controls. Stable SET knockdown (KD) was established by RNA interference in three KMT2A wild-type (wt) and four KMT2A-R leukemic cell lines. Gene and protein expression were analysed by RT-qPCR, ChiP, IP and western blot. RNA-seq and phospho-proteomics were employed to evaluate the effect of the SET-PP2A inhibitor FTY720 on global protein phosphorylation and gene expression. The cellular impact of FTY720 was evaluated by analysing proliferation, cell cycle and apoptosis in leukemic cell lines and by colony formation assay in two patient-derived xenograft (PDX). Results SET mRNA was found expressed in blasts from KMT2A-R-patients and in leukemic stem cells. SET protein interacted with both KMT2A wt and fusion proteins. Knockdown of SET inhibited the transcription of KMT2A target genes HOXA9 and HOXA10and abolished the self-renewal of KMT2A-R leukemic cells. Pharmacological inhibition of SET by FTY720 disrupted SET-PP2A interaction leading to cell cycle arrest, apoptosis and increased sensitivity to chemotherapy in KMT2A-R-leukemic models. Phospho-proteomic and western blot analyses revealed that FTY720 reduced the activity of kinases regulated by PP2A, including ERK1, GSK3b, ARKB, and led to degradation of MYC, supporting the hypothesis of a feedback loop among SET, PP2A and MYC. The RNA-seq indicated that FTY720 reduced the activity of signalling pathways implicated in gene transcription and it compromised the expression of several genes belonging to the KMT2A-R leukemia signature. Conclusions Taken together our results identify SET as a novel player in KMT2A-R leukemia and provide evidence that SET antagonism could serve as a novel strategy to treat this aggressive leukemia.

In response to tissue injury, within seconds the ultra-large glycoprotein von Willebrand factor (VWF) is released from endothelial storage organelles (Weibel−Palade bodies) into the lumen of the blood vasculature, where it leads to the recruitment of platelets. The marked size of VWF multimers represents an unprecedented burden on the secretory machinery of endothelial cells (ECs). ECs have evolved mechanisms to overcome this, most notably an actomyosin ring that forms, contracts, and squeezes out its unwieldy cargo. Inhibiting the formation or function of these structures represents a novel therapeutic target for thrombotic pathologies, although characterizing proteins associated with such a dynamic process has been challenging. We have combined APEX2 proximity labeling with an innovative dual loss-of-function screen to identify proteins associated with actomyosin ring function. We show that p21 activated kinase 2 (PAK2) recruits septin hetero-oligomers, a molecular interaction that forms a ring around exocytic sites. This cascade of events controls actomyosin ring function, aiding efficient exocytic release. Genetic or pharmacological inhibition of PAK2 or septins led to inefficient release of VWF and a failure to form platelet-catching strings. This new molecular mechanism offers additional therapeutic targets for the control of thrombotic disease and is highly relevant to other secretory systems that employ exocytic actomyosin machinery.

Introduction: We recently discovered C57BL/6N mice develop dilated cardiomyopathy (DCM), while the closely related C57BL/6J do not. We identified a variant (Chr8:85365179A>T, c.-5T>A) in myosin light chain kinase 3 ( Mylk3 ) in the C57BL/6N substrain that abolishes translation of the predominant transcript (ENSMUST00000034133). MYLK3 phosphorylates many sarcomere proteins, in particular myosin light chain 2 (MYL2), vital for cardiac development and function. Hypothesis: The MYLK3 variant is sufficient to drive the development of DCM. Results: We generated a novel isogenic mouse line carrying the Mylk3 c.-5T>A variant on the C57BL/6J background (C57BL/6J Mylk3.6N ). We found symptoms of DCM in male mutant mice at just 4 weeks old, with increased left ventricular diameter and volume, thinning of the posterior wall and a reduced ejection fraction. These data suggest the Mylk3 variant alone is sufficient to cause DCM. Surprisingly, we found no difference in phosphorylation of myosin light chain 2 (MYL2) by Phos-Tag® blotting at any age. This was unexpected, as previous reports in Mylk3 -/- mice found complete loss of MYL2 phosphorylation with a similar profile of DCM. However, in our mouse model, loss of MYLK3 is caused by a single point mutation in exon 1 which ablates the primary isoform (ENSMUST00000034133; Q3UIZ8) but western blotting with a C-terminal antibody revealed expression of a shorter isoform that is unaffected by the variant due to a distinct N-terminus (ENSMUST00000122452; D3Z630). There was no difference between the isoforms in their ability to phosphorylate MYL2 suggesting the shorter isoform can compensate. MYLK3 may also interact with ACTN2 and phosphorylate TNNI3. We found no difference in the expression or localisation of ACTN2 in our tissues, although co-IP in cells showed the primary isoform binds more readily to ACTN2, however, there was a significant reduction in TNNI3 S23/24 phosphorylation at all ages in C57BL/6J Mylk3.6N mice. This could affect cross-bridge cycling kinetics by altering the function of TNNI3 within the tropomyosin/troponin complex. Conclusions: Loss of full-length MYLK3 causes DCM via a MYL2-independent mechanism, possibly due to lack of phosphorylation of alternative target(s), such as TNNI3.

Introduction: The C57BL/6J and C57BL/6N are two of the most commonly used mouse strains in biological research. We recently discovered C57BL/6N males develop a modest dilated cardiomyopathy (DCM) by 12 months, which is absent from the closely related C57BL/6J. We identified a variant (Chr8:85365179A>T, c.-5T>A) in myosin light chain kinase 3 ( Mylk3 ) in the 5’ UTR of the primary isoform resident to C57BL/6N substrains, which abolishes translation of this isoform. Myosin light chain 2 (MYL2) is the primary target of MYLK3, and its phosphorylation is vital for survival. Hypothesis: Loss of MYLK3 expression alters phosphorylation of key cardiac proteins. Results: Surprisingly, we found no difference in MYL2 phosphorylation by western blot between C57BL/6N and C57BL/6J hearts, at any of the reported target residues. Phosphoproteomics corroborated this, but highlighted significantly different phosphorylation at a number of phosphosites in other proteins linked to cardiomyopathies and cardiac development, particularly a reduction of a number of residues in ACTN2. We also detected peptide sequences that mapped to the C-terminus and middle of MYLK3 in the C57BL/6N. Western blotting with a C-terminal specific antibody revealed expression of a shorter isoform, present at lower levels in both strains, that is unaffected by the variant due to a distinct N-terminus. Using this antibody in IHC showed similar spatial expression of MYLK3 in hearts from both strains, localizing to the Z-disc, although the expression was much lower in the C57BL/6N presumed to be due to lack of the more abundant full-length isoform. Using immunoprecipitation, we found both isoforms bound to MYL2 in vivo and in vitro , and both were capable of phosphorylating MYL2 in vitro. However, the shorter isoform was not as readily bound to alpha-actinin-2 (ACTN2), a key sarcomeric protein that is reported to bind full-length MYLK3 isoform by its N-terminus. Conclusions: This data indicates loss of full-length MYLK3 causes DCM via a MYL2-independent mechanism, possibly due to lack of phosphorylation of alternative target(s), such as ACTN2. The expression of the shorter MYLK3 isoform in C57BL/6N mice may explain the persistence of MYL2 phosphorylation in these mice and enable their survival.

Methodologies for proteomics and metabolomics are providing an unprecedented wealth of insights into cancer molecular biology. Although different techniques for proteomics and metabolomics exist, molecular snapshots in cancer metabolism and alterations in the proteome are mainly possible due to advancements in state-of-the-art mass spectrometry technologies. In this chapter, we describe examples of how proteo-metabolomic approaches are contributing to our understanding of the molecular biology of cancer progression, signalling, survival mechanisms, angiogenesis, and metastasis. We also provide an overview of the translational information (including biomarkers) and clinically relevant insights that proteomics and metabolomics strategies may be able to deliver, despite limitations and technical challenges that still exist. A better understanding of cancer progression and an improvement of clinical outcomes will benefit from precision medicine initiatives, in which appropriate application of proteo-metabolomic methods are key for their success.

Proper partitioning of homologous chromosomes during meiosis relies on the coordinated execution of multiple interconnected events: Homologs must locate, recognize, and align with their correct pairing partners. Further, homolog pairing must be coupled to assembly of the synaptonemal complex (SC), a meiosis-specific tripartite structure that maintains stable associations between the axes of aligned homologs and regulates formation of crossovers between their DNA molecules to create linkages that enable their segregation. Here, we identify HAL-3 (Homolog Alignment 3) as an important player in coordinating these key events during Caenorhabditis elegans meiosis. HAL-3, and the previously identified HAL-2, are interacting and interdependent components of a protein complex that localizes to the nucleoplasm of germ cells. hal-3 (or hal-2) mutants exhibit multiple meiotic prophase defects including failure to establish homolog pairing, inappropriate loading of SC subunits onto unpaired chromosome axes, and premature loss of synapsis checkpoint protein PCH-2. Further, loss of hal function results in misregulation of the subcellular localization and activity of Polo-like kinases (PLK-1 and PLK-2), which dynamically localize to different defined subnuclear sites during wild-type prophase progression to regulate distinct cellular events. Moreover, loss of PLK-2 activity partially restores tripartite SC structure in a hal mutant background, suggesting that the defect in pairwise SC assembly in hal mutants reflects inappropriate PLK activity. Together, our data support a model in which the nucleoplasmic HAL-2/HAL-3 protein complex constrains both localization and activity of meiotic Polo-like kinases, thereby preventing premature interaction with stage-inappropriate targets.

Significance Bone marrow failure (BMF) is an inherited life-threatening condition characterized by defective hematopoiesis, developmental abnormalities, and predisposition to cancer. BMF caused by ERCC6L2 mutations is considered to be a genome instability syndrome, because DNA repair is compromised in patient cells. In this study, we report BMF cases with biallelic disease-causing variants and provide evidence from patients’ cells that transcription deficiency can explain the genome instability. Specifically, we demonstrate that ERCC6L2 participates in RNA polymerase II-mediated transcription via interaction with DNA-dependent protein kinase (DNA-PK) and resolves DNA–RNA hybrids (R loops). Collectively, our data point to a causal mechanism in BMF in which patients with ERCC6L2 mutations are defective in the repair of transcription-associated DNA damage., Biallelic variants in the ERCC excision repair 6 like 2 gene (ERCC6L2) are known to cause bone marrow failure (BMF) due to defects in DNA repair and mitochondrial function. Here, we report on eight cases of BMF from five families harboring biallelic variants in ERCC6L2, two of whom present with myelodysplasia. We confirm that ERCC6L2 patients’ lymphoblastoid cell lines (LCLs) are hypersensitive to DNA-damaging agents that specifically activate the transcription coupled nucleotide excision repair (TCNER) pathway. Interestingly, patients’ LCLs are also hypersensitive to transcription inhibitors that interfere with RNA polymerase II (RNA Pol II) and display an abnormal delay in transcription recovery. Using affinity-based mass spectrometry we found that ERCC6L2 interacts with DNA-dependent protein kinase (DNA-PK), a regulatory component of the RNA Pol II transcription complex. Chromatin immunoprecipitation PCR studies revealed ERCC6L2 occupancy on gene bodies along with RNA Pol II and DNA-PK. Patients’ LCLs fail to terminate transcript elongation accurately upon DNA damage and display a significant increase in nuclear DNA–RNA hybrids (R loops). Collectively, we conclude that ERCC6L2 is involved in regulating RNA Pol II-mediated transcription via its interaction with DNA-PK to resolve R loops and minimize transcription-associated genome instability. The inherited BMF syndrome caused by biallelic variants in ERCC6L2 can be considered as a primary transcription deficiency rather than a DNA repair defect.

Cancer‐associated fibroblasts (CAFs) are considered the most abundant type of stromal cells in pancreatic ductal adenocarcinoma (PDAC), playing a critical role in tumour progression and chemoresistance; however, a druggable target on CAFs has not yet been identified. Here we report that focal adhesion kinase (FAK) activity (evaluated based on 397 tyrosine phosphorylation level) in CAFs is highly increased compared to its activity in fibroblasts from healthy pancreas. Fibroblastic FAK activity is an independent prognostic marker for disease‐free and overall survival of PDAC patients (cohort of 120 PDAC samples). Genetic inactivation of FAK within fibroblasts (FAK kinase‐dead, KD) reduces fibrosis and immunosuppressive cell number within primary tumours and dramatically decreases tumour spread. FAK pharmacologic or genetic inactivation reduces fibroblast migration/invasion, decreases extracellular matrix (ECM) expression and deposition by CAFs, modifies ECM track generation and negatively impacts M2 macrophage polarization and migration. Thus, FAK activity within CAFs appears as an independent PDAC prognostic marker and a druggable driver of tumour cell invasion., Understanding how cancer‐associated fibroblasts (CAFs) promote PDAC progression is of major interest given the poor prognosis of patients. This study identifies a druggable key regulator of CAF‐induced tumour cell metastasis and a prognostic factor: the protein Focal Adhesion Kinase (FAK).