PAK1 Activation Research Articles

Developing p21-activated kinase 1 (PAK1) activators to treat hypertrophic cardiomyopathy (HCM)

Abstract Despite significant progress in comprehending the genetic and metabolic underpinnings of cardiomyocyte dysfunction, there is still a pressing need for targeted treatments to address hypertrophy and progressive remodelling (e.g., fibrosis), seen in HCM1 . PAK1, a regulator of ion channels and myofilaments in cardiomyocytes, has shown promise in curbing pathological hypertrophy2 . Our hypothesis centres on the potential therapeutic benefits of pharmacologically activating PAK1 in managing hypertrophy and adverse remodelling in HCM. First, we conducted virtual screening and kinase assays to identify potent small molecule PAK1 activators, which significantly increased PAK1 activity by 3 to 5-fold, with an EC50 range between 0.5 and 2.5 μΜ. We then evaluated these compounds on cellular hypertrophy in an in vitro model using neonatal rat cardiomyocytes exposed to isoprenaline (ISO). Among those, JB2020A not only prevented ISO-induced hypertrophy, but also reversed pre-existing cellular hypertrophy induced by ISO 24 hours earlier. Subsequently, we assessed the therapeutic potential of JB2020A in a well-characterised HCM mouse model (i.e., Actc1E99K) 3 , known for its rapid disease progression. We initiated a six-week oral treatment regime at 4 weeks of age, dosing at 10 mg/kg per day. Echocardiography and fibrosis evaluation confirmed that JB2020A led to a significant reduction in cardiac hypertrophy and fibrosis with preserved contractile function. Western blotting analysis revealed increased PAK1 phosphorylation and activation in JB2020A-treated E99K hearts, along with a reduction in pro-apoptotic CHOP expression and an upregulation of protective endoplasmic reticulum (ER) response (e.g., ATF4 and Xbp1), indicating an amelioration of ER stress in HCM. These findings underscore the therapeutic potential of small molecule PAK1 activators as a novel approach for addressing hypertrophy and progressive remodelling in HCM.

Dose-dependent effects of silver nanoparticles on cell death modes in mouse blastocysts induced via endoplasmic reticulum stress and mitochondrial apoptosis.

In view of the rapidly expanding medical and commercial applications of silver nanoparticles (AgNPs), their potential health risks and environmental effects are a significant growing concern. Earlier research by our group uncovered the embryotoxic potential of AgNPs, showing detrimental impacts of these nanoparticles on both pre- and post-implantation embryonic development. In the current study, we showed that low (50-100μM) and high (200-400μM) dose ranges of AgNPs trigger distinct cell death programs affecting mouse embryo development and further explored the underlying mechanisms. Treatment with low concentrations of AgNPs (50-100μM) triggered ROS generation, in turn, inducing mitochondria-dependent apoptosis, and ultimately, harmful effects on embryo implantation, post-implantation development, and fetal development. Notably, high concentrations of AgNPs (200-400μM) evoked more high-level ROS generation and endoplasmic reticulum (ER) stress-mediated necrosis. Interestingly, pre-incubation with Trolox, a strong antioxidant, reduced ROS generation in the group treated with 200-400μM AgNPs to the level induced by 50-100μM AgNPs, resulting in switching of the cell death mode from necrosis to apoptosis and a significant improvement in the impairment of embryonic development. Our findings additionally indicate that activation of PAK2 is a crucial step in AgNP-triggered apoptosis and sequent detrimental effects on embryonic development. Based on the collective results, we propose that the levels of ROS generated by AgNP treatment of embryos serve as a critical regulator of cell death type, leading to differential degrees of damage to embryo implantation, post-implantation development and fetal development through triggering apoptosis, necrosis or other cell death signaling cascades.

The expression of congenital Shoc2 variants induces AKT-dependent crosstalk activation of the ERK1/2 pathway.

The Shoc2 scaffold protein is crucial in transmitting signals within the Epidermal Growth Factor Receptor (EGFR)-mediated Extracellular signal-Regulated Kinase (ERK1/2) pathway. While the significance of Shoc2 in this pathway is well-established, the precise mechanisms through which Shoc2 governs signal transmission remain to be fully elucidated. Hereditary variants in Shoc2 are responsible for Noonan Syndrome with Loose anagen Hair (NSLH). However, due to the absence of known enzymatic activity in Shoc2, directly assessing how these variants affect its function is challenging. ERK1/2 phosphorylation is used as a primary parameter of Shoc2 function, but the impact of Shoc2 mutants on the pathway activation is unclear. This study investigates how the NSLH-associated Shoc2 variants influence EGFR signals in the context of the ERK1/2 and AKT downstream signaling pathways. We show that when the ERK1/2 pathway is a primary signaling pathway activated downstream of EGFR, Shoc2 variants cannot upregulate ERK1/2 phosphorylation to the level of the WT Shoc2. Yet, when the AKT and ERK1/2 pathways were activated, in cells expressing Shoc2 variants, ERK1/2 phosphorylation was higher than in cells expressing WT Shoc2. In cells expressing the Shoc2 NSLH mutants, we found that the AKT signaling pathway triggers the PAK activation, followed by phosphorylation of Raf-1/MEK1/2 and activation of the ERK1/2 signaling axis. Hence, our studies reveal a previously unrecognized feedback regulation downstream of the EGFR and provide additional evidence for the role of Shoc2 as a "gatekeeper" in controlling the selection of downstream effectors within the EGFR signaling network.

Kinase activities in pancreatic ductal adenocarcinoma with prognostic and therapeutic avenues.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a limited number of known driver mutations but considerable cancer cell heterogeneity. Phosphoproteomics provides a direct read-out of aberrant signaling and the resultant clinically relevant phenotype. Mass spectrometry (MS)-based proteomics and phosphoproteomics were applied to 42 PDAC tumors. Data encompassed over 19 936 phosphoserine or phosphothreonine (pS/T; in 5412 phosphoproteins) and 1208 phosphotyrosine (pY; in 501 phosphoproteins) sites and a total of 3756 proteins. Proteome data identified three distinct subtypes with tumor intrinsic and stromal features. Subsequently, three phospho-subtypes were apparent: two tumor intrinsic (Phos1/2) and one stromal (Phos3), resembling known PDAC molecular subtypes. Kinase activity was analyzed by the Integrative iNferred Kinase Activity (INKA) scoring. Phospho-subtypes displayed differential phosphorylation signals and kinase activity, such as FGR and GSK3 activation in Phos1, SRC kinase family and EPHA2 in Phos2, and EGFR, INSR, MET, ABL1, HIPK1, JAK, and PRKCD in Phos3. Kinase activity analysis of an external PDAC cohort supported our findings and underscored the importance of PI3K/AKT and ERK pathways, among others. Interestingly, unfavorable patient prognosis correlated with higher RTK, PAK2, STK10, and CDK7 activity and high proliferation, whereas long survival was associated with MYLK and PTK6 activity, which was previously unknown. Subtype-associated activity profiles can guide therapeutic combination approaches in tumor and stroma-enriched tissues, and emphasize the critical role of parallel signaling pathways. In addition, kinase activity profiling identifies potential disease markers with prognostic significance.

Circ_0023990 Promotes the Proliferation, Invasion, and Glycolysis of Esophageal Squamous Cell Carcinoma Cells Via Targeting miR-6884-5p/PAK1 Axis.

Circular RNAs are emerging players in human cancers, including esophageal squamous cell carcinoma (ESCC). Herein, we assessed the expression level of circ_0023990 and explored the molecular mechanisms of circ_0023990 in ESCC. circ_0023990, miR-6884-5p, and PAK1 expressions in ESCC tissues and cells were detected by quantitative real-time polymerase chain reaction and western blot. ESCC cells were transfected with different constructs to alter the expression of circ_0023990, miR-6884-5p, and PAK1. The effect of circ_0023990 on the proliferation, invasion, and glycolysis of ESCC cells was observed. The interaction between circ_0023990 and miR-6884-5p and between miR-6884-5p and PAK1 were explored. A mouse model of ESCC was established to study the in vivo effect of circ_0023990 knockdown on tumor formation.The expression levels of circ_0023990 was upregulated in ESCC tissues and cells. Inhibiting circ_0023990 suppressed the proliferation, invasion, and glycolysis of ESCC cells. circ_0023990 might target miR-6884-5p and consequently modulate the expression and activity of PAK1. Knockdown of circ_0023990 led to significantly reduced tumor volume and weight in mice with ESCC.These findings overall suggest an oncogenic role of circ_0023990 in ESCC. Future research is warranted to confirm the expression pattern and clinical significance of circ_0023990 in ESCC.

Towards Understanding the Development of Breast Cancer: The Role of RhoJ in the Obesity Microenvironment.

Obesity is a growing pandemic with an increasing risk of inducing different cancer types, including breast cancer. Adipose tissue is proposed to be a major player in the initiation and progression of breast cancer in obese people. However, the mechanistic link between adipogenicity and tumorigenicity in breast tissues is poorly understood. We used in vitro and in vivo approaches to investigate the mechanistic relationship between obesity and the onset and progression of breast cancer. In obesity, adipose tissue expansion and remodeling are associated with increased inflammatory mediator's release and anti-inflammatory mediators' reduction.. In order to mimic the obesity micro-environment, we cultured cells in an enriched pro-inflammatory cytokine medium to which we added a low concentration of beneficial adipokines. Epithelial cells exposed to the obesity micro-environment were phenotypically transformed into mesenchymal-like cells, characterized by an increase in different mesenchymal markers and the acquisition of the major hallmarks of cancerous cells; these include sustained DNA damage, the activation of the ATR-Chk2 pathway, an increase in proliferation rate, cell invasion, and resistance to conventional chemotherapy. Transcriptomic analysis revealed that several genes, including RhoJ, CCL7, and MMP9, acted as potential major players in the observed phenomenon. The transcriptomics findings were confirmed in vitro using qRT-PCR and in vivo using high-fat-diet-fed mice. Our data suggests RhoJ as a potential novel molecular driver of tumor development in breast tissues and a mediator of cell resistance to conventional chemotherapy through PAK1 activation. These data propose that RhoJ is a potential target for therapeutic interventions in obese breast cancer patients.

Functional interactions between neurofibromatosis tumor suppressors underlie Schwann cell tumor de-differentiation and treatment resistance

Schwann cell tumors are the most common cancers of the peripheral nervous system and can arise in patients with neurofibromatosis type-1 (NF-1) or neurofibromatosis type-2 (NF-2). Functional interactions between NF1 and NF2 and broader mechanisms underlying malignant transformation of the Schwann lineage are unclear. Here we integrate bulk and single-cell genomics, biochemistry, and pharmacology across human samples, cell lines, and mouse allografts to identify cellular de-differentiation mechanisms driving malignant transformation and treatment resistance. We find DNA methylation groups of Schwann cell tumors can be distinguished by differentiation programs that correlate with response to the MEK inhibitor selumetinib. Functional genomic screening in NF1-mutant tumor cells reveals NF2 loss and PAK activation underlie selumetinib resistance, and we find that concurrent MEK and PAK inhibition is effective in vivo. These data support a de-differentiation paradigm underlying malignant transformation and treatment resistance of Schwann cell tumors and elucidate a functional link between NF1 and NF2.

The Splicing-Associated Network PAK1-Clk-SRRM1 Is a Critical Vulnerability to Overcome Chemoresistance in Acute Myeloid Leukemia

In Acute Myeloid Leukemia (AML), distinguishing between chemoresistance driver mechanisms and cell plasticity in response to chemotherapy is challenging due to their interconnectedness. To address this issue, we generated an MLL-AF9-driven syngeneic mouse model of AML, which we evolved to develop resistance to a front-line chemotherapy regimen by repetitive exposure to a maximally-tolerated combination of cytarabine and doxorubicin. An RNA sequencing (RNAseq)-based profiling of the chemoresistant (Chemo R) AML subpopulation and its naive (Chemo N) counterpart revealed a significant upregulation in the expression of 326 genes in Chemo R cells which were markedly enriched in gene sets related to splicing/mRNA processing. Notably, single-sample GSEA analysis of relapsed AML samples from the BEAT-AML cohort supported these findings, as they were enriched for the upregulation of genes in our chemoresistance gene signature. A combined in vivo functional screening endeavor using a pool of 2098 shRNAs targeting these upregulated genes and a motif discovery analysis performed on the set of dysregulated transcripts in Chemo R cells identified three candidate hits, Srrm1, Sf3b3, and Prpf8, which scored as chemotherapy-specific dependencies and were found to interact with central regulators of alternative splicing ( Figure 1A). Silencing these three candidate genes in our chemoresistant mouse model and the human AML cell lines, MOLM-14, MV4-11, and U937, evolved to become resistant to the combination of cytarabine and daunorubicin, revealed that the splicing regulator, SRRM1, is a critical vulnerability of chemoresistant leukemic cells. An orthogonal phospho-proteomic approach identified that PAK1 and CLK kinases are regulators of SRRM1 in chemoresistant AML cells, through the phosphorylation of multiple serine and threonine phosphosites in its arginine-rich domain. Human and murine chemoresistant cells were found to be markedly more sensitive to inhibitors of PAKs (FRAX597) and CLKs (TG003 and ML167) compared to their naive counterparts, an effect which was even further enhanced by treatment with cytarabine and daunorubicin. Notably, a whole-exome sequencing pipeline at our institution revealed a clonal variant of uncertain significance (c.1429G>T p.(Ala477Ser)) in PAK1 in an AML patient at relapse. Absence of this variant at diagnosis suggested its association with chemotherapy resistance. Biochemical and cellular assays confirmed that the A477S variant increased PAK1 activation as well as chemotherapy resistance in AML cell lines. This effect could be counteracted by suppressing SRRM1, indicating that PAK1 activation in relapsed AML confers increased vulnerability to SRRM1 suppression. Simultaneous suppression of Clk1 and Pak1 or Clk4 and Pak1 using validated shRNAs and combinations of FRAX597 and TG003, or FRAX597 and ML167 synergized to reduce the growth and the colony-forming capacity of chemoresistant AML cells and sensitized them to chemotherapy. These observations were supported by the RNAseq-based estimate of the inclusion ratios of alternative exons and introns which showed that the combination of PAK and CLK inhibitors phenocopied partially the effects of SRRM1 silencing on splicing perturbations. In preclinical studies, the combined treatment improved overall mouse survival and reduced disease burden in our chemoresistant AML mouse models compared to single-drug treatments ( Figure 1B). Moreover, AML patient cells that were primarily refractory or from a post chemotherapy relapse (n=21) exhibited a higher ex vivo sensitivity to the FRAX597 + TG003 combination in comparison with chemosensitive patients at diagnosis (n=28). Moreover, in NSG-S mice transplanted with matched samples from the same AML patient at diagnosis and relapse post-chemotherapy, combined PAK1 and CLK inhibition more effectively reduced disease progression in animals transplanted with the relapse sample than in those engrafted with the sample taken at diagnosis. In conclusion, our study advances the rationale that combined inhibition of PAK1 and CLK kinases effectively curtails malignant cell splicing adaptation to chemotherapy by affecting SRRM1 function. Combined PAK1 and CLK inhibitors either concomitant with or sequential to chemotherapy may offer greater clinical benefit and significantly improve AML patients' long-term outcomes.

Defining a Targetable Leukemia Intrinsic Dependency on Noncanonical PI3Kgamma Signaling

There are overlapping clinical and pathologic characteristics between poor prognosis subsets of acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN), but common dependencies are ill-defined. We hypothesized that identifying novel shared targets and biomarkers could elucidate biology and nominate new therapies for both malignancies. We performed genome-wide CRISPR interference in BPDCN cells and identified 327 dependency genes, which were then analyzed informatically and functionally in AML, ALL, and BPDCN cells and patient-derived xenografts (PDXs). We defined a leukemia subset characterized by PIK3R5 activation and sensitivity to phosphoinositide 3-kinase gamma (PI3Kγ) inhibition via a noncanonical downstream pathway. The PI3Kγ complex includes an enzymatic subunit p110γ encoded by PIK3CG and a regulatory subunit p101 encoded by PIK3R5. Intriguingly, despite elevation of both p110γ and p101 proteins in this leukemia subset, only the PIK3R5 mRNA is overexpressed. To investigate this disconnection, we measured protein half-life and found that p110γ stability is positively regulated by PIK3R5/p101, associated with reduced p110γ ubiquitination. This suggested that increased expression of PIK3R5/p101 leads to posttranslational p110γ elevation by protecting it from ubiquitin proteasome-mediated degradation. To identify the upstream activator of PIK3R5, we analyzed RNA-seq data in >200 leukemia PDXs and found that AMLs with activated PIK3R5 are enriched for an innate immune response signature (IIRS). Supporting this model and indicating potential clinical relevance, analysis of TCGA AML patients revealed that the IIRS, which includes PIK3R5, was associated with M4/M5 monocytic AML and poor prognosis, independent of age, cytogenetics, or somatic mutations. Furthermore, in cells with low baseline expression, PIK3R5 was induced by the Toll-like receptor agonist resiquimod, showing that PIK3R5 is activated by inflammatory signaling and consistent with a monocytic/dendritic lineage association. By integrated analysis of ATAC-seq and ChIP-seq in PIK3R5 high vs low leukemias, as well as gene dependency scores, we identified the transcription factor PU.1 as responsible for PIK3R5 activation. Treatment with resiquimod enhanced the interaction between PU.1 and the PIK3R5 promoter, while depletion of PU.1 nullified the ability of resiquimod to activate PIK3R5. To elucidate molecular events downstream of p101/PI3Kγ, we studied consequences of pathway inactivation using PIK3R5 interference or the PI3Kγ-selective inhibitor eganelisib. Surprisingly, there was no effect of PI3Kγ inactivation on AKT phosphorylation or canonical PI3K-AKT-mTOR mRNA targets. Therefore, we performed phosphoproteomics and found that PAK1 S144, a phosphorylation site not previously associated with PI3K signaling, was suppressed by PIK3R5/PI3Kγ inhibition ( panel a). Selective inhibitors of PAK1, but not AKT, replicated the effect of eganelisib. Furthermore, introduction of constitutively active PAK1, but not inactive PAK1, eliminated sensitivity of leukemias to eganelisib, supporting the functional relationship between PAK1 phosphorylation and PI3Kγ dependency. PI3Kγ inhibitors are being tested in solid tumor trials to repolarize macrophages and potentiate immune checkpoint blockade, but their cancer cell-intrinsic role has not been explored. To assess PI3Kγ inhibition in vivo, we first performed intradermal xenografting assays using BPDCN derived from a patient's skin tumor. Eganelisib achieved comparable efficacy to cytarabine chemotherapy and the combination of eganelisib and cytarabine exerted significant synergy (combination index=0.63, p<0.0001). Next, we measured the effect of PI3Kγ inhibition on survival in disseminated PDX models. As predicted, single agent eganelisib significantly prolonged survival in AML, ALL, and BPDCN with high PIK3R5, while having no benefit in cases with low PIK3R5. To our surprise, the combination of eganelisib/cytarabine synergistically increased survival in all cases, regardless of baseline PIK3R5 ( panel b). We conclude that inflammatory signaling-activated PIK3R5 is a biomarker for sensitivity to PI3Kγ inhibition via an unappreciated PI3Kγ-PAK1 axis. Synergy with cytarabine unmasked eganelisib sensitivity in additional leukemias, expanding the therapeutic potential of targeting PI3Kγ.

A PAK1 Mutational Hotspot Within the Regulatory CRIPaK Domain is Associated With Severe Neurodevelopmental Disorders in Children

BackgroundP-21-activated kinases (PAKs) are protein serine/threonine kinases, part of the RAS/mitogen-activated protein kinase pathway. PAK1 is highly expressed in the central nervous system and crucially involved in neuronal migration and brain developmental processes. Recently, de novo heterozygous missense variants in PAK1 have been identified as an ultrarare cause of pediatric neurodevelopmental disorders. MethodsWe report a series of children affected with postnatal macrocephaly, neurodevelopmental impairment, and drug-resistant epilepsy. Repeated electroencephalographic (EEG) and video-EEG evaluations were performed over a two- to 10-year period during follow-up to delineate electroclinical histories. Genetic sequencing studies and computational evaluation of the identified variants were performed in our patient cohort. ResultsWe identified by whole-exome sequencing three novel de novo variants in PAK1 (NM_001128620: c.427A>G, p.Met143Val; c.428T>C, p.Met143Thr; c.428T>A, p.Met143Lys) as the underlying cause of the disease in our families. The three variants affected the same highly conserved Met143 residue within the cysteine-rich inhibitor of PAK1 (CRIPaK) domain, which was identified before as a PAK1 inhibitor target. Computational studies suggested a defective autoinhibition presumably due to impaired PAK1 autoregulation as a result of the recurrent substitution. ConclusionsWe delineated the electroclinical phenotypes of PAK1-related neurological disorders and highlight a novel mutational hotspot that may involve defective autoinhibition of the PAK1 protein. The three novel variants affecting the same hotspot residue within the CRIPaK domain highlight potentially impaired PAK1-CRIPaK interaction as a novel disease mechanism. These findings shed light on possible future treatments targeted at the CRIPaK domain, to modulate PAK1 activity and function.

Identification of a novel PAK1/HDAC6 dual inhibitor ZMF-23 that triggers tubulin-stathmin regulated cell death in triple negative breast cancer

Triple-negative breast cancer (TNBC) is the most poorly treated subtype of breast cancer, and targeting the heterogeneity of TNBC has emerged as a fascinating therapeutic strategy. In this study, we propose for the first time that dual-targeting PAK1 and HDAC6 is a promising novel strategy for TNBC treatment due to their essential roles in the regulation of energy metabolism and epigenetic modification. We discovered a novel dual-targeting PAK1/HDAC6 inhibitor, 6 - (2-(cyclopropylamino) - 6 - (2,4-dichlorophenyl) - 7 - oxopyrido [2,3-d] pyrimidin - 8 (7H) -yl) - N-hydroxyhexanamide (ZMF-23), which presented profound inhibitory activity against PAK1 and HDAC6 and robust antiproliferative potency in MDA-MB-231 cells. In addition, SPR and CETSA assay demonstrated the targeted binding of ZMF-23 with PAK1/HDAC6. Mechanically, ZMF-23 strongly inhibited the cellular PAK1 and HDAC6 activity, impeded PAK1 and HDAC6 regulated aerobic glycolysis and migration. By RNA-seq analysis, ZMF-23 was found to induce TNF-α-regulated necroptosis, which further enhanced apoptosis. Additionally, ZMF-23 triggered PAK1-tubulin/HDAC6-Stathmin regulated microtubule structure changes, which further induced the G2/M cycle arrest. Moreover, prominent anti-proliferative effect of ZMF-23 was confirmed in the TNBC xenograft zebrafish and mouse model via PAK1 and HDAC6 inhibition. Collectively, ZMF-23 is a novel dual PAK1/HDAC6 inhibitor with TNBC treatment potential.

The Evaluation of Rac1 Signaling as a Potential Therapeutic Target of Alzheimer's Disease.

The Small GTPase Rac1 is critical for various fundamental cellular processes, including cognitive functions. The cyclical activation and inactivation of Rac1, mediated by Rac guanine nucleotide exchange factors (RacGEFs) and Rac GTPase-activating proteins (RacGAPs), respectively, are essential for activating intracellular signaling pathways and controlling cellular processes. We have recently shown that the Alzheimer's disease (AD) therapeutic drug donepezil activates the Rac1-PAK pathway in the nucleus accumbens (NAc) for enhanced aversive learning. Also, PAK activation itself in the NAc enhances aversive learning. As aversive learning allows short-term preliminary AD drug screening, here we tested whether sustained Rac1 activation by RacGAP inhibition can be used as an AD therapeutic strategy for improving AD-learning deficits based on aversive learning. We found that the RacGAP domain of breakpoint cluster region protein (Bcr) (Bcr-GAP) efficiently inhibited Rac1 activity in a membrane ruffling assay. We also found that, in striatal/accumbal primary neurons, Bcr knockdown by microRNA mimic-expressing adeno-associated virus (AAV-miRNA mimic) activated Rac1-PAK signaling, while Bcr-GAP-expressing AAV inactivated it. Furthermore, conditional knockdown of Bcr in the NAc of wild-type adult mice enhanced aversive learning, while Bcr-GAP expression in the NAc inhibited it. The findings indicate that Rac1 activation by RacGAP inhibition enhances aversive learning, implying the AD therapeutic potential of Rac1 signaling.

A missense variant at the RAC1-PAK1 binding site of RAC1 inactivates downstream signaling in VACTERL association

RAC1 at 7p22.1 encodes a RAC family small GTPase that regulates actin cytoskeleton organization and intracellular signaling pathways. Pathogenic RAC1 variants result in developmental delay and multiple anomalies. Here, exome sequencing identified a rare de novo RAC1 variant [NM_018890.4:c.118T > C p.(Tyr40His)] in a male patient. Fetal ultrasonography indicated the patient to have multiple anomalies, including persistent left superior vena cava, total anomalous pulmonary venous return, esophageal atresia, scoliosis, and right-hand polydactyly. After birth, craniofacial dysmorphism and esophagobronchial fistula were confirmed and VACTERL association was suspected. One day after birth, the patient died of respiratory failure caused by tracheal aplasia type III. The molecular mechanisms of pathogenic RAC1 variants remain largely unclear; therefore, we biochemically examined the pathophysiological significance of RAC1-p.Tyr40His by focusing on the best characterized downstream effector of RAC1, PAK1, which activates Hedgehog signaling. RAC1-p.Tyr40His interacted minimally with PAK1, and did not enable PAK1 activation. Variants in the RAC1 Switch II region consistently activate downstream signals, whereas the p.Tyr40His variant at the RAC1-PAK1 binding site and adjacent to the Switch I region may deactivate the signals. It is important to accumulate data from individuals with different RAC1 variants to gain a full understanding of their varied clinical presentations.

The role of P21-activated kinase (Pak1) in sinus node function

Sinoatrial node (SAN) dysfunction (SND) and atrial arrhythmia frequently occur simultaneously with a hazard ratio of 4.2 for new onset atrial fibrillation (AF) in SND patients. In the atrial muscle attenuated activity of p21-activated kinase 1 (Pak1) increases the risk for AF by enhancing NADPH oxidase 2 dependent production of reactive oxygen species (ROS). However, the role of Pak1 dependent ROS regulation in SAN function has not yet been determined. We hypothesize that Pak1 activity maintains SAN activity by regulating the expression of the hyperpolarization activated cyclic nucleotide gated cation channel (HCN).To determine Pak1 dependent changes in heart rate (HR) regulation we quantified the intrinsic sinus rhythm in wild type (WT) and Pak1 deficient (Pak1−/−) mice of both sexes in vivo and in isolated Langendorff perfused hearts. Pak1−/− hearts displayed an attenuated HR in vivo after autonomic blockage and in isolated hearts. The contribution of the Ca2+ clock to pacemaker activity remained unchanged, but Ivabradine (3 μM), a blocker of HCN channels that are a membrane clock component, eliminated the differences in SAN activity between WT and Pak1−/− hearts. Reduced HCN4 expression was confirmed in Pak1−/− right atria. The reduced HCN activity in Pak1−/− could be rescued by class II HDAC inhibition (LMK235), ROS scavenging (TEMPOL) or attenuation of Extracellular Signal-Regulated Kinase (ERK) 1/2 activity (SCH772984). No sex specific differences in Pak1 dependent SAN regulation were determined.Our results establish Pak1 as a class II HDAC regulator and a potential therapeutic target to attenuate SAN bradycardia and AF susceptibility.

Abstract 1586: The potential therapeutic effect of the Rac/Cdc42 inhibitor MBQ-167 in glioblastomain vitro

Abstract Glioblastoma is a highly aggressive astrocytoma known to form the most lethal type of primary brain tumor. Therapeutic options are limited because 94% of diagnosed patients survive less than five years. Glioblastoma malignancy is attributed to uncontrolled cellular proliferation, increased angiogenesis, apoptosis resistance, and local invasion. The Homologous small GTPases of the Rho family, Rac1 and Cdc42, are critical regulators of the invasion and migration of glioblastoma cells. Previous studies have found a correlation between the high expression of Rac and CDC42 and the highly invasive characteristics of GBM in vivo. Our group characterized the small molecule compound MBQ-167 as a potent dual inhibitor of Rac and Cdc42 in breast and pancreatic cancer and have shown its utility in cell and mouse models of metastasis. In this project, we tested the hypothesis MBQ-167 is also a valid therapy option for glioblastoma. The T98G glioblastoma cell line was treated with varying concentrations (62 nM to 1000 nM) of MBQ-167 for 24 hrs, and effects on Rac/PAK activation (at 24 hours) was determined through active GTP bound Rac pulldowns, cell viability (at 48 and 96 hours) through MTT assays, and cellular migration (at 12, 24, and 48 hours) through wound healing assays. Results show that 250 nM of MBQ-167 inhibits Rac and PAK activation by 50% as well as cell viability at both time points (48 and 96 hours). MBQ-167 (150 nM-500 nM) also blocked glioblastoma cell migration at 12, 24, and 48 hours. Taken together, these results confirm that MBQ-167 is a viable inhibitor in glioblastoma. Citation Format: Derealise Garcia Almedina, Nilmary Grafals-Ruiz, Suranganie Dharmawardhane, Anamaris Torres-Sanchez. The potential therapeutic effect of the Rac/Cdc42 inhibitor MBQ-167 in glioblastomain vitro [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 1586.

Pharmacological Inhibition of p-21 Activated Kinase (PAK) Restores Impaired Neurite Outgrowth and Remodeling in a Cellular Model of Down Syndrome.

Down syndrome (DS) is characterized by the trisomy of chromosome 21 and by cognitive deficits that have been related to neuronal morphological alterations in humans, as well as in animal models. The gene encoding for amyloid precursor protein (APP) is present in autosome 21, and its overexpression in DS has been linked to neuronal dysfunction, cognitive deficit, and Alzheimer's disease-like dementia. In particular, the neuronal ability to extend processes and branching is affected. Current evidence suggests that APP could also regulate neurite growth through its role in the actin cytoskeleton, in part by influencing p21-activated kinase (PAK) activity. The latter effect is carried out by an increased abundance of the caspase cleavage-released carboxy-terminal C31 fragment. In this work, using a neuronal cell line named CTb, which derived from the cerebral cortex of a trisomy 16 mouse, an animal model of human DS, we observed an overexpression of APP, elevated caspase activity, augmented cleavage of the C-terminal fragment of APP, and increased PAK1 phosphorylation. Morphometric analyses showed that inhibition of PAK1 activity with FRAX486 increased the average length of the neurites, the number of crossings per Sholl ring, the formation of new processes, and stimulated the loss of processes. Considering our results, we propose that PAK hyperphosphorylation impairs neurite outgrowth and remodeling in the cellular model of DS, and therefore we suggest that PAK1 may be a potential pharmacological target.

Abstract P5-02-36: Proteogenomic profiling of fresh frozen core biopsies from CALGB 40601

Abstract Background: Targeted therapy for HER2+ breast cancer has significantly improved outcomes for this aggressive subtype. However, a subset of patients do not achieve pathological complete response (pCR). In CALGB 40601, a randomized Phase III Trial for neoadjuvant treatment of HER2+ primary breast cancer with Paclitaxel (T: taxane) combined with HER2 antibody therapy (H: Herceptin/Trastuzumab), the small molecule inhibitor Lapatinib (L), or the antibody-inhibitor combination, pCR frequency was 56% for the combination (THL arm), 46% for Trastuzumab (TH arm), and 32% for Lapatinib (TL arm, closed early because of lower efficacy) (PMID: 26527775). While a recent publication reports relapse-free survival (RFS), overall survival (OS), and RNA-based gene expression signatures that can predict pCR (PMID: 33095682), understanding the proteogenomic landscape of treatment response should facilitate identification of alternative and therapeutically tractable protein targets for treatment-resistant tumors. Methods: Microscaled proteogenomic profiling (PMID: 31988290) was performed on treatment-naïve, flash-frozen core needle biopsies from the CALGB 40601 trial obtained from the Alliance for Clinical Trials in Oncology tissue bank. Multi-omics profiling included whole-exome sequencing (WES), RNA-sequencing, and mass spectroscopy-based proteomics and phosphoproteomics from one or two cores from each patient. Results: Eighty baseline core biopsies from 54 patients, including 22 patients from the THL arm, 24 from the TH arm, and 8 from the TL arm, from the CALGB 40601 tissue archive were of sufficient quality to yield genomics, transcriptomics, and/or proteomics profiling data. The frequency of pCR for profiled samples was representative of the overall trial cohort. Linear models were employed to identify baseline determinants of pCR for each arm and to assess differences in genes associated with response between the TH and THL arms. Pathways associated with RNA processing, translation, and the proteasome were elevated in pCR tumors in TH and THL arms, while cell cycle, DNA replication and repair pathways were higher in pCR only in the THL arm. While enrichment of similar pathways was observed in pCR in the transcriptome, the proteome specifically showed enrichment of pathways associated with extracellular matrix and EMT in non-pCR in the THL but not the TH arm. In particular, “EMT”, “ECM-receptor interaction”, and “extracellular structure organization” constituted the most enriched pathways and GO terms that were higher in non-pCR than in pCR tumors from the combination arm (THL) in the proteomics data despite showing no enrichment in the transcriptomics data. Driving this pathway enrichment were several collagens and matrix metalloproteinases that were significantly elevated in non-pCR tumors at the protein but not the RNA level. Finally, kinase target enrichment of differential phosphorylation sites suggested that the activity of PAK1, a regulator of cytoskeletal remodeling, is elevated in non-pCR tumors from the THL arm (p=0.006), but not the TH arm (p=0.69). Conclusion: Proteogenomic analysis of archival HER2+ breast cancer core biopsies provides opportunities for identifying proteins and phosphorylation sites in treatment-naive tumors that are associated with pCR to neoadjuvant Paclitaxel/anti-HER2 therapy. Notably, proteomic but not transcriptomic data showed that ECM and EMT pathways were elevated in non-pCR tumors; thus, signatures encompassing these pathways may serve as biomarkers for aggressive HER2+ breast cancer that is more likely to evade treatment. Non-pCR tumors in the THL arm were also marked by elevated levels of PAK1 target phosphorylation sites, suggesting that this kinase may be a potential therapeutic target in HER2+ breast cancer that is refractory to combination anti-HER2 therapy. Citation Format: Eric J. Jaehnig, Aranzazu Fernandez-Martinez, Tanmayi Vashist, Matthew V. Holt, LaTerrica Williams, Jonathan Lei, Beom-Jun Kim, Yongchao Dou, Viktoriya Korchina, Richard Gibbs, Donna Muzny, Harshavardhan Doddapaneni, Henry Rodriguez, Ana Robles, Tara Hiltke, DR Mani, Michael Gillette, Terry Hyslop, Yujia Wen, Linda McCart, George Miles, Steven Carr, Bing Zhang, Shankha Satpathy, Matthew Ellis, Meenakshi Anurag. Proteogenomic profiling of fresh frozen core biopsies from CALGB 40601 [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 P5-02-36.

Rac is required for the survival of cortical neurons.

Rac1, a member of small Rho GTPases, is involved in diverse cellular processes in neuronal cells. Rac1 plays especially important roles during development, and its roles have been extensively studied using Rac1-deficient mice. Rac3, a close homolog of Rac1, is ubiquitously expressed in the nervous system and may therefore compensate for Rac1 in Rac1-deficient cells. Exploration of the roles of Rac in neurons may therefore be difficult. We thus deleted both Rac1 and Rac3 in cortical neurons. Rac-deficient cerebral cortices formed slightly hypoplastic but almost normally layered structures at birth, but cortical neurons underwent apoptosis soon after birth. Rac-deficient cortical neurons had poor survivability and there was reduction in the length and the number of neurites in vitro. Activation of Pak1, a downstream effector of Rac, in Rac-deficient cortical neurons rescued the survivability in vitro. Pak1-activated Rac-deficient neurons had numerous dendrites, but no axons. Restoration of p35, a regulator of Cdk5, partly rescued the survivability of Rac-deficient neurons both in vitro and in vivo. Expression of p35 also partly rescued the length and the number of neurites in Rac-deficient neurons in vitro. Rac was shown to be indispensable for the survival of cortical neurons, and Pak1 and Cdk5/p35 work as downstream effectors of Rac to promote neuronal survival.

TrkA expression directs the anti-neoplastic activity of MLK3 inhibitors in triple-negative breast cancer.

Mixed Lineage Kinase 3 (MLK3) is a viable target for neoplastic diseases; however, it is unclear whether its activators or inhibitors can act as anti-neoplastic agents. We reported that the MLK3 kinase activity was higher in triple-negative (TNBC) than in hormone receptor-positive human breast tumors, where estrogen inhibited MLK3 kinase activity and provided a survival advantage to ER+ breast cancer cells. Herein, we show that in TNBC, the higher MLK3 kinase activity paradoxically promotes cancer cell survival. Knockdown of MLK3 or MLK3 inhibitors, CEP-1347 and URMC-099, attenuated tumorigenesis of TNBC cell line and Patient-Derived (PDX) xenografts. The MLK3 kinase inhibitors decreased both the expression and activation of MLK3, PAK1, and NF-kB protein and caused cell death in TNBC breast xenografts. RNA-seq analysis identified several genes downregulated by MLK3 inhibition, and the NGF/TrkA MAPK pathway was significantly enriched in tumors sensitive to growth inhibition by MLK3 inhibitors. The TNBC cell line unresponsive to kinase inhibitor had substantially lower TrkA, and overexpression of TrkA restored the sensitivity to MLK3 inhibition. These results suggest that the functions of MLK3 in breast cancer cells depend on downstream targets in TNBC tumors expressing TrkA, and MLK3 kinase inhibition may provide a novel targeted therapy.

Neutrophil Extracellular Traps Delay Diabetic Wound Healing by Inducing Endothelial-to-Mesenchymal Transition via the Hippo pathway.

Diabetic foot ulcers (DFUs) are among the most frequent complications of diabetes with significant morbidity and mortality. Diabetes can trigger neutrophils to undergo histone citrullination by protein arginine deiminase 4 (encoded by Padi4 in mice) and release neutrophil extracellular traps (NETs). The specific mechanism of NETs-mediated wound healing impairment in diabetes remains unknown. In this study, we show neutrophils are more susceptible to NETosis in diabetic wound environments. Via in vitro experiments and in vivo models of wound healing using wide-type and Padi4 -/- mice, we demonstrate NETs can induce the activation of PAK2 via the membrane receptor TLR-9. Then PAK2 phosphorylates the intracellular protein Merlin/NF2 to inhibit the Hippo-YAP pathway. YAP binds to transcription factor SMAD2 and translocates from the cytoplasm into the nucleus to promote endothelial-to-mesenchymal transition (EndMT), which ultimately impedes angiogenesis and delays wound healing. Suppression of the Merlin/YAP/SMAD2 pathway can attenuate NET-induced EndMT. Inhibition of NETosis accelerates wound healing by reducing EndMT and promoting angiogenesis. Cumulatively, these data suggest NETosis delays diabetic wound healing by inducing EndMT via the Hippo-YAP pathway. Increased understanding of the molecular mechanism that regulates NETosis and EndMT will be of considerable value for providing cellular targets amenable to therapeutic intervention for DFUs.