PAK Proteins Research Articles

Abstract 2629: Single and combined inhibition of PAK and Hippo pathway in NF2-deficient schwannoma

Abstract Neurofibromatosis 2 (NF2) Syndrome is a rare, but devastating genetic condition that leads to non-cancerous tumors of the nervous system. NF2 patients are likely to develop Schwannomas on the eighth cranial nerve that lead to progressive deafness and spinal cord compression as well as meningiomas and peripheral nerve tumors. Effective treatment for these patients is lacking, with risky surgery being the most reliable approach and pharmacotherapies having inadequate results. The hallmark mutation of this condition is inactivation of NF2, which codes for the protein Merlin. Merlin plays several roles in the cell, two of the most consequential being inhibition of PAK1 and activation of the Hippo tumor suppressor pathway. However, drugs targeting these pathways have not yet been investigated in NF2-deficient Schwannoma. In this study, we explored the use of YAP-TEAD inhibitors as well as the combination of Hippo family and Group I PAK inhibition in NF2-deficient Schwannoma cells. We used several Schwannoma cells lines of human, mouse, and rat origin as well as a human Schwann cell line with NF2 knocked out via CRISPR compared to its wild-type control line. We investigated the role of YAP, the main effector of the Hippo pathway, and PAK1/2 in NF2 Schwannoma cells via genetic and pharmacologic inhibition. We also combined YAP-TEAD inhibitors with several PAK family inhibitors to induce a synergistic reduction in cell viability and proliferation as well as an increase in apoptosis. We found that genetic inhibition of both YAP and Group I PAK proteins led to partial reduction in Schwannoma growth. Using several YAP-TEAD binding inhibitors (TED-347, NSC682769, IK-930), Group I PAK inhibitors (FRAX-1036, G555) and PAK1-specific inhibitor (NVS-1-PAK1) we found that YAP-TEAD binding inhibition combined with Group I PAK inhibition has the greatest reduction on Schwannoma cell viability, proliferation, and increase in cell death. Taken together, our findings indicate that both the Hippo pathway and PAK proteins may be viable targets for Schwannoma therapies. Furthermore, combining the two may lead to a synergistic effect and further prevent growth of NF2-deficient Schwannomas. Citation Format: Dorothy Benton, Jonathan Chernoff. Single and combined inhibition of PAK and Hippo pathway in NF2-deficient schwannoma [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 2629.

PAK1 regulates ATXN1 levels providing an opportunity to modify its toxicity in spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 (SCA1) is caused by the expansion of a trinucleotide repeat that encodes a polyglutamine tract in ataxin-1 (ATXN1). The expanded polyglutamine in ATXN1 increases the protein's stability and results in its accumulation and toxicity. Previous studies have demonstrated that decreasing ATXN1 levels ameliorates SCA1 phenotypes and pathology in mouse models. We rationalized that reducing ATXN1 levels through pharmacological inhibition of its modulators could provide a therapeutic avenue for SCA1. Here, through a forward genetic screen in Drosophila we identified, p21-activated kinase 3 (Pak3) as a modulator of ATXN1 levels. Loss-of-function of fly Pak3 or Pak1, whose mammalian homologs belong to Group I of PAK proteins, reduces ATXN1 levels, and accordingly, improves disease pathology in a Drosophila model of SCA1. Knockdown of PAK1 potently reduces ATXN1 levels in mammalian cells independent of the well-characterized S776 phosphorylation site (known to stabilize ATXN1) thus revealing a novel molecular pathway that regulates ATXN1 levels. Furthermore, pharmacological inhibition of PAKs decreases ATXN1 levels in a mouse model of SCA1. To explore the potential of using PAK inhibitors in combination therapy, we combined the pharmacological inhibition of PAK with MSK1, a previously identified modulator of ATXN1, and examined their effects on ATXN1 levels. We found that inhibition of both pathways results in an additive decrease in ATXN1 levels. Together, this study identifies PAK signaling as a distinct molecular pathway that regulates ATXN1 levels and presents a promising opportunity to pursue for developing potential therapeutics for SCA1.

P21-activated kinase 4 regulates HIF-1α translation in cancer cells

The p21-activated kinases (Paks) interact with Rac/Cdc42 GTPases to regulate the actin cytoskeleton as well as various signaling pathways. Although activation of Paks in many human cancers is known to mediate cancer progression, the role of Pak proteins in hypoxia is poorly understood. In this study, we found that both Pak1 and Pak4 are highly expressed in HeLa cervical cancer cells, but only Pak4 knockdown attenuates expression of hypoxia-inducible factor-1α (HIF-1α) in hypoxia. We further discovered that Pak4 regulates HIF-1α translation via the Akt-mTOR-4E-BP1 pathway under hypoxic conditions. These results support a novel connection between HIF-1α and Pak4 in hypoxic cancer cells, and provide insights into mechanisms whereby tumors respond to and thrive under oxygen-deficient conditions.

Therapeutic Potential of Targeting PAK Signaling.

The therapeutic potential of targeting p21-Activated Kinases (PAK1 - 6) for the treatment of cancer has recently gained traction in the biotech industry. Many pharmaceutically-viable ATP competitive inhibitors have been through different stages of pre-clinical development with only a single compound evaluated in human trails (PF-3758309). The best studied functional roles of PAK proteins are control of cell adhesion and migration. PAK proteins are known downstream effectors of Ras signaling with PAK expression elevated in cancer (pancreatic, colon, breast, lung and other solid tumors). In addition altered PAK expression is a confirmed driver of this disease, especially in tumors harboring oncogenic Ras. However, there are very few examples of gain-of-function PAK mutations, as a majority of the cancer types have elevated PAK expression due to gene amplification or transcriptional modifications. There is a substantial number of known substrates affected by this aberrant PAK activity. One particular substrate, β-catenin, has garnered interest given its importance in both normal and cancer cell development. These data place PAK proteins between two major signaling pathways in cancer (Ras and β -catenin), making therapeutic targeting of PAKs an intriguing approach for the treatment of a broad array of oncological malignancies.

Abstract 4039: Inhibition of Pak4 blocks growth of breast cancer cells

Abstract The p21-activated kinase family of proteins promotes cell survival and plays an important role in cell proliferation, cell cycle regulation and cell shape determination. There are six mammalian PAK proteins which can be subdivided into two groups by sequence homology and mode of activation- Group A Paks consisting of Pak 1, 2 and 3 and Group B Paks consisting of Pak 4, 5 and 6. There is a growing list of evidence that PAK proteins are overexpressed in many cancer types, including colon, lung, and breast cancer. PAK4 is frequently overproduced in breast cancer, including Her2 positive and triple negative breast cancers, while it is expressed at low levels in normal mammary tissue. Our preliminary evidence showed that untransformed mouse mammary epithelial cells have low levels of PAK4 that become elevated when these cells are transformed by Her2 overexpression. Furthermore, PAK4 overexpressed in mammary epithelial cells leads to oncogenic transformation and tumorigenesis in mice. We hypothesize that PAK4 plays an important role in promoting mammary tumorigenesis, and that it can serve as an effective drug target for breast cancer treatment. To test this hypothesis, we have analyzed two orally available PAK4 Allosteric Modulators (PAM; KPT-8752 and KPT-9274), which reduce the steady state PAK4 protein level in cancer cells. We observed that when we treat breast cancer cells with KPT-8752, we severely block the cell growth. KPT-8752, is effective both in Her2 positive and triple negative breast cancer cells. Our results indicate that Pak4 is an important regulator of breast cancer cell growth. Experiments studying the effects of PAMs (KPT8752 and KPT9274) on tumorigenesis in animal models of breast cancer are ongoing. Citation Format: Chetan K. Rane, William Senapedis, Erkan Baloglu, Sharon Shacham, Audrey G. Minden. Inhibition of Pak4 blocks growth of breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4039. doi:10.1158/1538-7445.AM2015-4039

Abstract 5404: PAK4 allosteric modulators (PAMs) repress the Wnt/β-catenin signaling pathway and tumor growth

Abstract Wnt/β-catenin signaling is a ubiquitous pathway conserved throughout evolution and plays a role in embryonic and cancer development. This pathway has proven to be difficult to target therapeutically. Another intractable target is oncogenic Ras which is highly mutated in many cancer types, including pancreatic, colon and lung. An available target at the intersection of both of these genes is p21-Activated Kinase 4 (PAK4). PAK4 (1 of 6 PAK proteins) that is downstream of the Ras oncogene and a direct kinase that stabilizes transcriptional activity of β-catenin. Therefore therapeutically targeting PAK4 in cancer could be beneficial in a broad range of cancer types. PAK4 Allosteric Modulator (PAM) represents a novel and selective class of compounds that inhibit PAK4 allosterically. Methods: Flow cytometry and CellTiter AQueous One assay (MTS) were used to determine compound effects on cell cycle distribution and proliferation. CCLE, COSMIC and other databases were used for bioinformatics analysis of mutations in cancer genes. Deep sequencing and qPCR were used to analyze mRNA expression profiles. Proteomic platforms - KinomeScan, PTScan (Cell Signaling) and immunoblots were used to study phosphorylation signature of whole cell proteins and total protein steady state levels. Results: We identified a selective, orally bioavailable small molecule PAM, KPT-9274, which demonstrated anti-tumor activity in a variety of cancer cell lines (IC50 values from 0.005 - 1 μM). Bioinformatics revealed that sensitivity to KPT-9274 was directly correlated with mutations in APC or N-ras and inversely correlated with mutations in β-catenin, K-ras, or PI3K. KPT-9274 and other PAMs reduced phosphorylation and steady state levels of PAK4 protein while reducing Phospho-S675 and total β-catenin, Wnt5, Phospho- and total LRP6, Dvl2, Phospho-Akt, and Phospho-ERK. PAMs also reduced β-catenin transcriptional activity (i.e. cyclin D1, MM7, and S100A4). PAMs arrested cancer cell cycle at the G1 and G2 phases and induced apoptosis through Caspase and PARP cleavage. KPT-9274 (∼100 mg/kg BIDx5 orally) has demonstrated potent anti-tumor activity against hematological (Z-138, Molt-4, MM1S) and solid (MDA-MB-468 and Colo-205) xenograft models in mice. PAM-treated xenografts showed reduction of PAK4, β-catenin and cyclin D1 proteins. Conclusions: PAK4 represents a novel anti-cancer target at the crossroads of Ras and Wnt/β-catenin signaling. We have identified selective small molecule PAMs with anti-tumor activity both in vitro and in vivo. These allosteric modulators induce tumor cell growth arrest and apoptosis. Bioinformatics helped identify potential predictive markers in the Wnt signaling pathway while deep sequencing and proteiomics revealed possible PDn markers. Based on the in vitro and in vivo activity, KPT-9274 may be beneficial for the treatment of a wide variety of cancers and preclinical toxicology studies are ongoing. Citation Format: William T. Senapedis, Scott Donovan, Gali Golan, Dilara McCauley, Joel Ellis, Marsha Crochiere, Trinayan Kashyap, Boris Klebanov, Sharon Shacham, Yosef Landesman, Erkan Baloglu. PAK4 allosteric modulators (PAMs) repress the Wnt/β-catenin signaling pathway and tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5404. doi:10.1158/1538-7445.AM2015-5404

A Critical Role of GIT1 in Vertebrate and Invertebrate Brain Development.

GIT1, a multifunctional signaling adaptor protein, is implicated in the development of dendritic spines and neuronal synapses. GIT1 forms a signaling complex with PIX, RAC, and PAK proteins that is known to play important roles in brain development. Here we found that Git1-knockout (Git1-/-) mice show a microcephaly-like small brain phenotype, which appears to be caused by reduced neuronal size rather than number. Git1-/- mice also show decreased dendritic spine number without morphological alterations in the hippocampus. Behaviorally, Git1-/- mice show impaired motor coordination and learning and memory. In addition, adult dGit Drosophila mutants show decreased brain size and abnormal morphology of the mushroom body. These results suggest that GIT1 is important for brain development in both rodents and flies.

Anti-inflammatory property of propolis.

Dear Editor-in-Chief, The recent article by Weina Gao and colleagues, on July issue of this Journal, showed that Brazilian green propolis have a positive effect on innate and adaptive immunity in aged mice.(1) This role should be mainly attributed to flavonoids contained in propolis.(2) The Authors reported that Brazilian propolis had 189.12 mg/g total polyphenols, 98.46 mg/g flavonoids, 1.95 mg/g cinnamic acid and 23 mg/g artepillin-C.(1) Yet, the participation of any single molecule to the reported evidence in aged animals might have involved different spectra of activity and different molecular targets in promoting the beneficial action observed by the Authors.(1) Artepillin C, namely (E)-3-[4-hydroxy-3,5-bis(3-methylbut-2-enyl)phenyl]prop-2-enoic acid, is one of the major component in Brazilian propolis and recent reports have stressed its ability to block the serine/theonine protein kinase known as PAK-1.(3) PAK proteins are critical effectors that link the Rho family of GTPases to the cytoskeleton reorganization and nuclear signaling; These proteins serve as targets for the small GTP binding proteins Cdc42 and Rac and have been implicated in a wide range of biological activities, for example PAK1 regulates cell motility and morphology, furthermore the role of Cdc42 GTPases activating protein in aging has been recently reported.(4) The involvement of PAK1 in inflammatory and neuro-degenerative disorders suggests that this molecules is targeted in aging mechanism. Few natural PAK1-blockers such as rosmarinic acid, curcumin and caffeic acid extend the lifespan of the nematode Caenorhabditis elegans or fruit flies.(5) PAK1 promotes reproduction, whereas it inactivates HSP16.2 gene and shortens lifespan, as do also PI-3 kinase (AGE-1), mTOR, and insulin-like signalling (ILS) (Daf-2) in this nematode.(5) It is arguable that caffeic acid and particularly artepillin-C play a role in slowing aging cellular mechanism and synergistically exert an anti-inflammatory action with propolis flavonoids.(4) This suggestive speculation may appear contradictory when data from serum in aged mice, showing a marked increase in inflammatory cytokines on increasing Brazilian propolis assumption, are considered.(1) The Authors concluded that this evidence assessed the improvement in inflammatory response caused by propolis in aged mice.(1) This circumstance addresses the positive role exhibited by nature-derived phytochemicals on stressed, dysregulated, aged or infected (inflamed) cells. Therefore, while a wide literature exists reporting the anti-inflammatory role of flavonoids,(6) many data from clinics should suggest a pro-inflammatory role exerted by polyphenols in restoring health, prevent cancer onset and degenerative disorders.(7) Fundamentally, as like as in propolis, the most proper idea about the activity of nature-derived polyphenols and phenolic substances, should deal with the concept of immune modulation, depending on the cell nature and homeostatic balance, not merely an improvement given by inhibition or promotion coming from the molecular nature of these molecules. Propolis is widely used as an anti-inflammatory raw substance, expecially during allergy and airway inflammatory disorders. The interesting paper by Weina Gao et al., would suggest that in elderly people Brazilian propolis may ensure a health promoting action by activating the immune response.(1) This is formally true but literature reports many data with potentially misleading evidence about. A combination therapy (anti-inflammatory drugs plus propolis) in aged adults showed encouraging results(8) and, moreover, due to yet contradictory results from clinics,(9) respect to in vitro and animal evidence,(1,10) a more cautious, critical and less enthusiastic debate about this substance has to be taken into account in the next future. Looking forward to a kind reply from yours.

Coronin1 proteins dictate rac1 intracellular dynamics and cytoskeletal output.

Rac1 regulates lamellipodium formation, myosin II-dependent contractility, and focal adhesions during cell migration. While the spatiotemporal assembly of those processes is well characterized, the signaling mechanisms involved remain obscure. We report here that the cytoskeleton-related Coronin1A and -1B proteins control a myosin II inactivation-dependent step that dictates the intracellular dynamics and cytoskeletal output of active Rac1. This step is signaling-branch specific, since it affects the functional competence of active Rac1 only when forming complexes with downstream ArhGEF7 and Pak proteins in actomyosin-rich structures. The pathway is used by default unless Rac1 is actively rerouted away from the structures by upstream activators and signals from other Rho GTPases. These results indicate that Coronin1 proteins are at the center of a regulatory hub that coordinates Rac1 activation, effector exchange, and the F-actin organization state during cell signaling. Targeting this route could be useful to hamper migration of cancer cells harboring oncogenic RAC1 mutations.

Key Nodes In FLT3 Dependent Signaling Determine Growth and Survival Of Childhood Acute Lymphoblastic Leukemia

The balanced regulation of complex signaling networks plays an important role in cell proliferation, survival and apoptosis. Receptor tyrosine kinase (RTK) dependent signal transduction has been implicated in the pathogenesis of many malignancies including acute lymphoblastic leukemia (ALL) of childhood. Moreover, deregulated RTK activity was observed following targeted inhibition of constitutively active kinases or kinase-dependent pathways in a variety of malignancies, which conferred pharmacological resistance. In ALL, fms-like-tyrosine kinase 3 (FLT3) and platelet-derived growth factor receptor beta (PDGFRβ) are targeted for mutation (Roberts et al., 2012). However, in primary ALL the RTK dependent signaling state is poorly defined and the occurrence of relapse in the context of genotype-directed monotherapy regimens targeting RTKs, such as FLT3, underlines the need for an activity-based approach to RTK signaling in leukemia comprising the identification of critical downstream target proteins.To select for driver RTKs in ALL, we combined the analyses of RTK expression in primary ALL (n=102), ALL cell lines and normal hematopoietic cells with the characterization of ligand dependent cell proliferation as well as shRNA mediated RTK repression in vitro and in vivo. We observed aberrant RTK expression patterns in ALL cells compared to normal lymphoid progenitor and stem cell populations as well as mature T- and B-lymphocytes. RNA interference mediated repression of growth promoting RTKs FLT3 and PDGFRβ in primary ALL led to a loss or reduction of the affected cell population in vivo. To identify critical signaling nodes we performed a phosphoproteomic characterization by iTRAQ (isobaric tags for relative and absolute quantification)-based mass spectrometry of the signal transduction of selected driver RTKs in the corresponding primary ALL samples. Primary ALL cells were propagated in NSG mice after xenotransplantation, and regulated phosphoproteins were identified after ligand stimulation. Our network-directed approach to RTK signaling in ALL thus allowed for the identification of downstream signaling nodes implicated in aberrant RTK activity.We identified a total of 2241 phosphoproteins and observed a striking diversity of RTK driven signaling processes in primary ALL exhibiting only a marginal overlap between phosphoregulated proteins which illustrates the inter-individual heterogeneity and the challenge for non-combinatorial therapies. Despite a predominant receptor and cell type specific composition of potentiated signaling networks our phosphoproteomic analyses identified p21-activated protein kinase PAK2 as a novel key nodal point in FLT3 dependent signaling in ALL. The importance of PAK protein family members in the regulation of cell proliferation and survival and the emerging role of PAK proteins in the pathogenesis of a broad range of tumors suggests a hitherto unanticipated function in the malignant transformation of ALL and the signal transduction of FLT3 (Ye and Field, 2012).As a kinase PAK2 represents a druggable target and may be suited for combinatorial intervention strategies targeting FLT3 signaling in order to induce synthetic lethality. Inhibition of group I PAK kinases (PAK1, 2, 3) using the allosteric inhibitor IPA-3 and RNA interference mediated repression of PAK2 led to the loss of ALL cells due to an impaired cell proliferation and an increased apoptosis. Notably, PAK2 depleted ALL cells showed an elevated sensitivity towards pharmacological FLT3 inhibition which underlines the potential role of PAK2 as a novel target in ALL and the need for novel small molecule PAK inhibitors with higher specificity and improved applicability in vivo. Disclosures:No relevant conflicts of interest to declare.

The Rac GTPase effector p21-activated kinase is essential for hematopoietic stem/progenitor cell migration and engraftment

AbstractThe p21-activated kinases (Paks) are serine/threonine kinases that are major effectors of the Rho guanosine 5′\\x{2011}triphosphatase, Rac, and Cdc42. Rac and Cdc42 are known regulators of hematopoietic stem and progenitor cell (HSPC) function, however, a direct role for Paks in HSPCs has yet to be elucidated. Lin-Sca1+c-kit+ (LSK) cells from wild-type mice were transduced with retrovirus expressing Pak inhibitory domain (PID), a well-characterized inhibitor of Pak activation. Defects in marrow homing and in vitro cell migration, assembly of the actin cytoskeleton, proliferation, and survival were associated with engraftment failure of PID-LSK. The PID-LSK demonstrated decreased phosphorylation of extracellular signal-regulated kinase (ERK), whereas constitutive activation of ERK in these cells led to rescue of hematopoietic progenitor cell proliferation in vitro and partial rescue of Pak-deficient HSPC homing and engraftment in vivo. Using conditional knock-out mice, we demonstrate that among group A Paks, Pak2−/− HSPC show reduced homing to the bone marrow and altered cell shape similar to PID-LSK cells in vitro and are completely defective in HSPC engraftment. These data demonstrate that Pak proteins are key components of multiple engraftment-associated HSPC functions and play a direct role in activation of ERK in HSPCs, and that Pak2 is specifically essential for HSPC engraftment.

Redundant canonical and noncanonical Caenorhabditis elegans p21-activated kinase signaling governs distal tip cell migrations.

p21-activated kinases (Paks) are prominent mediators of Rac/Cdc42-dependent and -independent signaling and regulate signal transduction and cytoskeletal-based cell movements. We used the reproducible migrations of the Caenorhabditis elegans gonadal distal tip cells to show that two of the three nematode Pak proteins, MAX-2 and PAK-1, function redundantly in regulation of cell migration but are regulated by very different mechanisms. First, we suggest that MAX-2 requires CED-10/Rac function and thus functions canonically. Second, PIX-1 and GIT-1 function in the same role as PAK-1, and PAK-1 interaction with PIX-1 is required for PAK-1 activity; thus, PAK-1 functions noncanonically. The human Pak-Pix-Git complex is central to noncanonical Pak signaling and requires only modest Rac/CDC-42 input. Unlike the human complex, our results suggest that the C. elegans Pak-Pix-Git complex requires PAK-1 kinase domain activity. This study delineates signaling network relationships in this cell migration model, thus providing potential further mechanistic insights and an assessment of total Pak contribution to cell migration events.

EphB2 receptor forward signaling controls cortical growth cone collapse via Nck and Pak

EphB receptors and their ephrinB ligands transduce bidirectional signals that mediate contact-dependent axon guidance primarily by promoting growth cone repulsion. However, how EphB receptor-mediated forward signaling induces axonal repulsion remains poorly understood. Here, we identify Nck and Pak proteins as essential forward signaling components of EphB2-dependent growth cone collapse in cortical neurons. We show that kinase-active EphB2 binds to Pak and promotes growth cone repulsion via Pak kinase activity, Pak-Nck binding, RhoA signaling and endocytosis. However, Pak's function in this context appears to be independent of Rac/Cdc42-GTP, consistent with the absence of Rac-GTP production after ephrinB treatment of cortical neurons. Taken together, our findings suggest that ephrinB-activated EphB2 receptors recruit a novel Nck/Pak signaling complex to mediate repulsive cortical growth cone guidance, which may be relevant for EphB forward signaling-dependent axon guidance in vivo.

Pak A Proteins Are Essential for Hematopoietic Stem/Progenitor Cell (HSC/P) Engraftment Through Regulation of Signaling Pathways Controlling HSC/P Proliferation, Survival, Migration, and the Actin Cytoskeleton

Abstract 917Rho GTPases, including Rac, integrate multiple extracellular signals and play important regulatory roles in HSC/P functions such as engraftment, retention, migration, adhesion, proliferation, and survival (Gu et al. Science, 2003). Our studies now focus on identifying potential Rac downstream effector proteins important for normal HSC/P function(s). The p21-activated kinases (Pak) are serine/threonine kinases that interact with and are major downstream targets of Rac and Cdc42. There are six human Paks (Pak1–6), which are grouped based on homology into Group A (Pak 1–3) and Group B (4–6) Paks. Paks regulate cytoskeletal organization including stress fiber dissolution, lamellipodia formation and focal adhesion disassembly and mediate activation of MAPK pathways. To identify the possible role(s) of Pak proteins in engraftment, freshly isolated LSK cells from WT (CD45.1+/CD45.2+) BM were transduced with retrovirus containing the Pak Inhibitory Domain (PID), which inhibits Group A Pak protein function or empty vector control (Mieg3); both constructs co-express GFP. 1.0×105 GFP+ LSK+ cells were then isolated and co-transplanted with 5.0×105 BoyJ (CD45.1+) whole bone marrow (WBM) into lethally irradiated C57Bl/6J (CD45.2+) recipients. Percent chimerism was measured at 3- to 24- weeks post BMT. PID transduced LSK+ cells were incapable of contributing to recipient hematopoietic reconstitution (Table 1). To explore the underlying mechanism of this engraftment failure we performed in vivo homing assays and found a 4- and 16- fold decrease, respectively, in BM homing of PID transduced LSK+ vs controls at 12 and 48 hours (p<0.05, for both time points). Altered cell migration of LSK+ cells was confirmed by live imaging microscopy which showed a 4-fold decrease in overall cell displacement in SDF-1-stimulated directed migration in the PID-expressing LSK+ compared to controls and was associated with a two-fold increase in random cell migration of PID-transduced LSK+ cells in transwell migration assays. PID-expressing LSK+ cells also demonstrated abnormal lamellipodia associated with significant increases in both cell surface area and cell perimeter. Because cytoskeletal changes may be linked to alterations in cell growth, we next examined the effect of Pak inhibition on cell survival and proliferation. PID-expressing LSK+ cells had decreased proliferation (17.7% vs 36.8% of cells in S-phase, p<0.05) and increased apoptosis (48.1% vs 16.7% AnnexinV+ cells, p<0.05) when compared to controls, respectively. These phenotypic changes were associated with decreased pERK and pAKT in PID-expressing LSK+. To confirm the importance of Pak activation of these proteins in HSC/P, we performed experiments to rescue the observed engraftment defect by co-transducing PID or Mieg3 with a constitutively active-ERK (ca-MEK1) or ca-AKT. We found ca-MEK1, but not ca-AKT, was able to increase proliferation in vitro (% proliferating cells for PID + empty vector = 6.1% and PID+ ca-MEK1 = 9.5%; p<0.05) and partially but only transiently rescue Pak-deficient HSC/P engraftment (% donor cells for LSK+ transduced with: PID + empty vector =1.5%, PID + ca-MEK1 =15.8%, and PID + ca-AKT =0.5% at 3-weeks post-BMT; p<0.05 for empty vector vs ca-MEK1). Finally, to determine which PakA pathway is critical in HSC engraftment we studied Pak genetic knock-out cells. We found that Pak2Δ/Δ -but not Pak1−/− -cells resulted in a profound HSC/P engraftment defect (% Pak2Δ/Δ vs Pak2flox/flox and Pak1−/− vs Pak1wt/wt: 1.0% vs 26.5% and 35.8% vs 37.4%; p<0.05 and p=ns, respectively at 3-weeks). Taken together, these data suggest that Pak A proteins regulate multiple HSC/P functions and link Rac GTPases to actin cytoskeletal rearrangements, directed cell migration, and proliferation/survival of HSC/P during engraftment.TABLE 1:Percentage of GFP+ cells in peripheral blood of recipient mice at indicated time points post BMT3-weeks6-weeks10-weeks14-weeks24-weeksWT-Mieg331.6% (±6.69)27.1% (±8.6)34.6% (±15.0)43.2% (±16.3)22.2% (±10.7)WT-PID0.22 (±0.19)0.07% (0.06)0%0%0%**Data represent mean ± s.d., n=10 recipients per group, p<0.05 for all time points, two independent experiments. Disclosures:No relevant conflicts of interest to declare.

Protein Kinase D Regulates Cofilin Activity through p21-activated Kinase 4

Dynamic reorganization of the actin cytoskeleton at the leading edge is required for directed cell migration. Cofilin, a small actin-binding protein with F-actin severing activities, is a key enzyme initiating such actin remodeling processes. Cofilin activity is tightly regulated by phosphorylation and dephosphorylation events that are mediated by LIM kinase (LIMK) and the phosphatase slingshot (SSH), respectively. Protein kinase D (PKD) is a serine/threonine kinase that inhibits actin-driven directed cell migration by phosphorylation and inactivation of SSH. Here, we show that PKD can also regulate LIMK through direct phosphorylation and activation of its upstream kinase p21-activated kinase 4 (PAK4). Therefore, active PKD increases the net amount of phosphorylated inactive cofilin in cells through both pathways. The regulation of cofilin activity at multiple levels may explain the inhibitory effects of PKD on barbed end formation as well as on directed cell migration.

Group A Pak Proteins Are Essential for HSC/P Engraftment.

AbstractAbstract 2624Rho GTPases, including Rac, integrate multiple extracellular signals and play important regulatory roles in critical hematopoietic stem and progenitor (HSC/P) functions such as cell migration, adhesion, proliferation, and survival. Conditional deletion of Rac1 in the hematopoietic cells results in defects in proliferation and engraftment while Rac2 germline deletion leads to enhanced cell death and increased HSC/P mobilization, most likely resulting from decreased adhesion via altered integrin signalling (Gu et al. Science, 2003). Although, Rac1 and Rac2 proteins have been shown to regulate distinct biological processes, simultaneous deletion of both Rac1 and Rac2 reveal overlapping functions in HSC/P survival, mobilization, engraftment, and retention (Cancelas et al. Nat. Med., 2006). Our studies now focus on indentifying potential downstream effector proteins of Rac that are important for normal HSC/P function(s). The best characterized Rac effector proteins are the Pak protein kinases, including Group A (Pak1, Pak2, and Pak3) Paks. Pak proteins not only have an intrinsic kinase activity but also regulate actin-dependent functions. Previous studies have demonstrated inhibition of Rac in HSC/P was associated with reduced phosphorylation of Pak1 (Cancelas et al. Nat. Med., 2006). However, the exact role Pak proteins play in HSC/P function(s) has yet to be elucidated. To further identify the possible role(s) of Pak proteins in hematopoiesis, freshly isolated lineage-, Sca-1+; c-Kit+ (LSK) cells from BoyJ(CD45.1)/C57Bl6J(CD45.2) F1 donor mice were transduced with retroviral vectors containing the Pak Inhibitory Domain (PID), which inhibits Group A Pak protein function, or empty vector control (Mieg3); both constructs co-express GFP. 6.0×104 GFP+ CD45.1/CD45.2 LSK cells were then isolated and co-transplanted with 5.0×105 BoyJ (CD45.1) bone marrow (BM) into lethally irradiated C57Bl/6J (CD45.2) recipients. Percent chimerism was measured from 3-weeks up to 14-weeks post BM transplant. We found that PID transduced LSK cells were incapable of contributing to recipient hematopoietic reconstitution compared to Mieg3 transduced controls at 14 weeks post-transplant (0/9 PID transduced demonstrated >1% engraftment vs 9/9 Mieg3 transduced which had >10% engraftment, p<0.05). To explore the underlying mechanism of this engraftment failure, we cultured transduced LSK in vitro for up to 12 days and compared growth of GFP+ vs GFP- cells. The growth of PID transduced LSK was essentially identical to non-transduced cells for the initial 7 days of in vitro culture. However between day 7 and day 10, the PID LSK cells displayed decreased growth kinetics (Fig. 1). The reduced growth in vitro was correlated with decreased proliferation as measured by BrdU incorporation. There was no change in cell survival, as assessed by AnnexinV staining. Furthermore, LSK cells transduced with a dominant negative Pak construct (DN-Pak), a more general inhibitor of Pak activity, showed defects in cytoskeletal rearrangements after stimulation with the HSC/P chemoattract, SDF-1α. These data suggest that Pak proteins may be critical downstream effectors of Rac GTPases and defective Pak activation may mechanistically link the role of Rac GTPases in regulation of both cytoskeletal and proliferative functions of HSC/P during engraftment. [Display omitted] Disclosure:Sanchez-Aguilera:Children's Hospital Boston: Employment.

P21-activated kinase 5 inhibits camptothecin-induced apoptosis in colorectal carcinoma cells

p21-activated kinase 5 (PAK5) is a recently identified member of the group B PAK family. The PAK proteins are effectors of the small GTPase Cdc42 and Rac1 and are known to regulate cell motility and activate cell-survival signaling pathways. Especially, the mitochondrial localization of PAK5 is vital to its effects on apoptosis and cell survival. Previously, we demonstrated that PAK5 expression increased significantly during the malignant progression of colorectal carcinoma (CRC) and that PAK5 promoted CRC metastasis by regulating CRC cell adhesion and migration. In the present study, we aim to investigate the role of PAK5 in camptothecin-induced apoptosis and its potential mechanism of action. Our results showed that overexpression of PAK5 inhibited camptothecin-induced apoptosis by inhibiting the activity of caspase-8 in CRC cells. Accordingly, knockdown of PAK5 in LoVo cells resulted in increased apoptosis. Mechanistically, we found that PAK5 directly phosphorylated Bad on serine 112 and indirectly led to phosphorylation of serine 136 via the Akt pathway. In conclusion, our study revealed previously unappreciated inhibitory role of PAK5 in camptothecin-induced apoptosis, thus suggesting PAK5 as a novel therapeutic target in CRC.

Erbin and the NF2 Tumor Suppressor Merlin Cooperatively Regulate Cell-Type-Specific Activation of PAK2 by TGF-β

Transforming growth factor beta (TGF-beta) family ligands are pleotropic proteins with diverse cell-type-specific effects on growth and differentiation. For example, PAK2 activation is critical for the proliferative/profibrotic action of TGF-beta on mesenchymal cells, and yet it is not responsive to TGF-beta in epithelial cells. We therefore investigated the regulatory constraints that prevent inappropriate PAK2 activation in epithelial cultures. The results show that the epithelial-enriched protein Erbin controls the function of the NF2 tumor suppressor Merlin by determining the output of Merlin's physical interactions with active PAK2. Whereas mesenchymal TGF-beta signaling induces PAK2-mediated inhibition of Merlin function in the absence of Erbin, Erbin/Merlin complexes bind and inactivate GTPase-bound PAK2 in epithelia. These results not only identify Erbin as a key determinant of epithelial resistance to TGF-beta signaling, they also show that Erbin controls Merlin tumor suppressor function by switching the functional valence of PAK2 binding.

Spotlight

O'Sullivan et al., pp. 1930–1940 PAK proteins, a family of serine/threonine p21-activating kinases, are critical effectors that link RhoGTPases to cell proliferation and cytoskeleton reorganization. PAK1 is the best characterized member of the PAK family, which also includes PAK2, PAK3 and PAK4 proteins. PAK1 gene amplifications and increased protein expression have been associated with breast, ovarian and colorectal carcinomas. In this issue of IJC, researchers from the Cork Cancer Research Center in Ireland identify PAK1 as a potential new drug target in kidney cancer. Renal cell carcinoma arises from the renal tubules and is the most common form of kidney cancer in adults. Notoriously resistant to chemotherapy and radiotherapy, it has been treated with some success by immunotherapy. The identification of PAK1 adds the possibility of yet another treatment option for this highly refractory tumor. O'Sullivan and colleagues discovered that PAK1, but not PAK2 or PAK3, was markedly overexpressed in several renal cell lines. Overexpressed PAK1 was hyperphosphorylated at threonine 423, a residue recognized by the phosphoinositide-dependent kinase 1 (PDK1) and required for full kinase activity. Expression of PAK1 and phospho-PAK1 was confirmed in tumor samples and correlated well to tumor stages. Introduction of a dominant-negative form of PAK1 or RNAi-mediated knockdown of the protein in 786-0 renal cancer cell lines decreased cell proliferation and anchorage-independent cell growth in vitro. Importantly, expression of dominant-negative PAK1 prevented growth of renal tumors in athymic MF1 nu-nu mice. These data support the model that therapeutic inhibition of PAK1 could become a critical new component in the treatment of patients with renal cell carcinoma. Kanagawa et al., pp. 2013–2022 Colorectal cancer represents one the leading causes of cancer deaths in the western world. While early diagnosis and prevention have made a significant difference, 20% of patients present with inoperable cancer at diagnosis and 50% of patients that undergo a potentially curative surgical resection relapse. New targeted therapeutic strategies are therefore needed. There is growing evidence that immune mechanisms have a prognostic significance in colorectal carcinoma. In particular, the presence of a tumor lymphocytic infiltrate appears to be significant. In this issue of the IJC, Kanagawa and colleagues tested the effect of several chemokines in a murine model of preexisting CT26 colon carcinoma. Chemokines regulate leukocyte trafficking and infiltration at local sites and are attractive candidates for cancer immunotherapy. Mice carrying a CT26 tumor were injected at the tumor site with adenovirus vectors expressing individual chemokines. The authors report that expression of CCL17 induced tumor regression and generated specific immunity in rechallenge experiments. These effects of CCL17 were dependent both on the vector dose and the number of injections. CCL17 expression in tumors was associated with significant increases in the number of infiltrating macrophages and CD8 T cells at the tumor sites. These observations warrant further investigation of the potential immunotherapeutic role of CCL17 in colorectal cancer in humans. Lambert et al., pp. 2052–2059 In the past 50 years, impressive progress has been made in the treatment of cancers of the colon and rectum. Patient survival has increased for both cancers due to improved patient care. However, it remained unclear how such progress affected patient survival: were more patients “cured” or did the survival time of the “uncured” increase? Using data from the Finish Cancer Registry, Lambert and colleagues show that both are true. In the years from 1953 to 2003, cure fractions and median survival of the uncured group increased in both cancers. The cure fractions are now broadly similar, but median survival of the uncured remains higher for cancer of the rectum. A reason could be that metastatic disease has been the predominant cause of death in colon cancer patients whereas rectal cancer patients often experience local recurrence, which is slower and now accessible to treatment. Postoperative mortality decreased after the introduction of intensive care units during the late 1960s and early 1970s. Combination chemotherapy became available in the late 1980s and early 1990s, and recently, surgeons have started to resect metastases in the liver. These factors have likely promoted the constant improvement in patient care. However, survival rates for patients with rectal cancer have not increased further in the past 5 years. In addition, the cure rate of colon cancer, which experienced a steep linear increase for patients above 70 years, was less improved in patients younger than 50 years. These factors indicate that cancer patient survival might plateau rather than further increase unless new curative treatments are found.

The p21-activated Kinase 3 Implicated in Mental Retardation Regulates Spine Morphogenesis through a Cdc42-dependent Pathway

The p21-activated kinase 3 (PAK3) is one of the recently identified genes for which mutations lead to nonsyndromic mental retardation. PAK3 is implicated in dendritic spine morphogenesis and is a key regulator of synaptic functions. However, the underlying roles of PAK3 in these processes remain poorly understood. We report here that the three mutations R419X, A365E, and R67C, responsible for mental retardation have different effects on the biological functions of PAK3. The R419X and A365E mutations completely abrogate the kinase activity. The R67C mutation drastically decreases the binding of PAK3 to the small GTPase Cdc42 and impairs its subsequent activation by this GTPase. We also report that PAK3 binds significantly more Cdc42 than Rac1 and is selectively activated by endogenous Cdc42, suggesting that PAK3 is a specific effector of Cdc42. Interestingly, the expression of the three mutated proteins in hippocampal neurons affects spinogenesis differentially. Both kinase-dead mutants slightly decrease the number of spines but profoundly alter spine morphology, whereas expression of the R67C mutant drastically decreases spine density. These results demonstrate that the Cdc42/PAK3 is a key module in dendritic spine formation and synaptic plasticity.