Atypical protein kinase C (aPKC) isozymes are unique in the protein kinase C (PKC) superfamily in that they are not regulated by the lipid second messenger diacylglycerol. Whether a different second messenger acutely controls their function is unknown. Here we show that the lipid mediator, sphingosine 1-phosphate (S1P), controls the cellular activity of aPKC. Using a genetically-encoded reporter we designed, aPKC-specific C Kinase Activity Reporter (aCKAR), we demonstrate that intracellular S1P activates aPKC. Biochemical studies reveal that S1P directly binds to the kinase domain of aPKC to relieve autoinhibitory constraints. In silico studies identify potential binding sites on the kinase domain, one of which was validated biochemically. Lastly, functional studies reveal that S1P-dependent activation of aPKC suppresses apoptosis in HeLa cells. Taken together, our data reveal a previously undescribed molecular mechanism for controlling the cellular activity of atypical PKC and identify a new molecular target for S1P.

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Synthetic lethality occurs when a single gene alteration is compatible with cell viability, but an additional co-occurring genetic alteration leads to cell death. In the context of cancer therapy, synthetic lethality can occur through the inhibition of a target that is selectively essential to tumors harboring a specific genetic alteration. Gene paralog pairs represent one promising class of synthetic lethal cancer targets, wherein the function of one paralog is lost in tumor cells, rendering them dependent on the remaining paralog to carry out an essential cellular process. To identify essential gene paralog pairs as starting points for drug discovery programs, we mined publicly available CRISPR genetic loss-of-function data and associated molecular datasets collected across a diverse panel of cancer cell lines. We first identified pairs of gene paralogs where one paralog was essential in a subset of cell lines, and then filtered these genes based on function, known literature, enrichment in specific lineages and integration of external datasets. These efforts identified VPS4A as a synthetic lethal target in cancers harboring copy number loss of VPS4B. VPS4A and VPS4B are highly homologous AAA ATPases that carry out multiple essential cellular processes including nuclear membrane remodeling and endosomal membrane biogenesis. VPS4B loss occurs as a passenger deletion during loss of the tumor suppressors SMAD2 and SMAD4. Loss of VPS4B creates a genetic dependency on VPS4A to drive essential VPS4-dependent processes. VPS4B deletion occurs at a frequency of up to 3% in multiple solid tumor types including esophageal, head and neck, pancreatic and colorectal cancers. To further explore the potential of VPS4A as a therapeutic target in VPS4B-deleted tumors, we first validated the synthetic lethal relationship between VPS4A/B using isogenic cell line pairs. HCT116 cells with an engineered homozygous loss of VPS4B, but not wild-type HCT116 cells, showed profound cell kill in response to genetic silencing of VPS4A. Moreover, simultaneous siRNA-mediated knockdown of VPS4A and VPS4B resulted in cell death across a panel of cancer cell lines (e.g. H1975, Panc0403), while knockdown of either gene alone was compatible with cell viability. Encouraged by these results, we profiled several previously reported small-molecule inhibitors of VPS4A (e.g. DBeQ and MSC1094308) in a suite of biochemical assays. Notably, these molecules were inactive against VPS4A. We have discovered a novel series of VPS4A inhibitors and are advancing this inhibitor series through lead optimization. Potent, selective, and pharmacologically active VPS4A inhibitors are expected to be well tolerated and have strong single-agent activity in tumors bearing VPS4B homozygous deletions. Citation Format: Meredith Kuo, Jason Chen, Sacha Holland, Eugene Lurie, An-Angela Ngoc Van, Francesco Parlati, Tayna Santos, Eric Sjogren, Natalija Sotirovska, Susanne Steggerda, Andrew MacKinnon. Identification of novel VPS4A inhibitors for the treatment of VPS4B-deleted cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1816.

Gain‐of‐function fusion proteins have emerged as novel cancer drivers and potential therapeutic targets, exemplified by the successful targeting of the oncogenic Bcr‐Abl gene fusion that drives chronic myelogenous leukemia. Over 60 fusions of protein kinase C (PKC) family members with other genes have been identified in diverse cancers, accounting for the most abundant fusions in the AGC family of kinases. Given our recent findings that PKC isozymes generally function as tumor suppressors, we explored whether such fusions may provide another mechanism for loss of PKC function in cancer. Specifically, we addressed the cellular activity of fusions in which the C‐terminal catalytic domain of PKC was retained, but the N‐terminal regulatory domain was truncated by fusion to the N‐terminus of unrelated genes: these fusions are TANC2‐PRKCA, which encodes a protein containing the first 46 residues of the adaptor protein TANC2 fused to the N‐terminus of the C2 domain of PKCα, and GGA2‐PRKCB, which encodes a protein containing the first 158 residues of the Golgi trafficking protein GGA2 fused to the C1A domain of PKCβ. Overexpression in COS7 cells revealed that both fusion proteins are constitutively active as assessed by the FRET‐based C Kinase Activity Reporter (CKAR). This constitutive activity is not a result of the various proteins to which PKC is fused, but rather is a consequence of loss of the autoinhibitory pseudosubstrate from within the PKC regulatory moiety; constructs of PKC deleted in the pseudosubstrate are also constitutively active in cells. However, the fusion proteins are unstable as assessed by their accelerated turnover compared to wild‐type PKC following inhibition of protein synthesis. Because active PKC is in a degradation‐sensitive conformation, we reasoned that PKC fusion proteins would be too unstable for significant levels to accumulate in cancer cells. To test this, we used CRISPR/Cas9 to edit one allele of PKCα to express the TANC2‐PRKCA fusion observed in cancer. Western blot analysis of these clones revealed readily detectable PKCα from the intact allele but no detectable fusion protein, verifying that the PKC fusion is indeed too unstable to accumulate in cells. Thus, while the fusion proteins are rendered constitutively active due to a loss of autoinhibitory constraints, their inherent instability results in a dramatic reduction of their steady‐state levels, effectively making them loss‐of‐function. PKC fusion proteins therefore present another mechanism by which loss of PKC activity can occur in the context of cancer, supporting a tumor suppressive role for PKC.Support or Funding InformationThis work was supported by NIH R35 GM122523 (ACN). ANV, TRB, and CEA were supported by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology (T32 GM007752), and CEA was supported by an NSF Graduate Research Fellowship (DGE1144086).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Over 60 protein kinase C (PKC) fusion genes have been identified in a wide variety of cancers, making PKC the most frequently fused AGC kinase. Our laboratory previously revealed that cancer‐associated point mutations annotated for PKC family members are generally loss‐of‐function, with functional studies revealing a tumor suppressive role for PKC. Here, we sought to determine whether PKC fusions may also be loss‐of‐function. In our analysis, we characterized TANC2‐PRKCA, a fusion identified in lung squamous cell carcinoma that retains the catalytic domain of PKCα, and PRKCA‐CDH8, a fusion identified in breast cancer that retains the regulatory domain of PKCα. Overexpression of TANC2‐PKCα in cells showed that the fusion protein is constitutively active; however, it lacks phosphorylation at key sites required for the stability of the enzyme. Most importantly, without its amino‐terminal pseudosubstrate, the fusion protein is unable to adopt the autoinhibited, stable conformation of wild‐type PKC, rendering it markedly unstable. To determine whether the fusion protein could accumulate in cells given this instability, we utilized CRISPR/Cas9‐mediated gene editing to express TANC2‐PRKCA. Indeed, while the fusion mRNA was detected in the CRISPR‐edited clones, we were unable to detect the fusion protein. Thus, although the fusion is rendered constitutively active through loss of autoinhibition, its subsequent instability prevents its accumulation in cells, making it paradoxically loss‐of‐function. In contrast to fusions that retain the PKC catalytic domain, fusions that retain the PKC regulatory domain are intrinsically loss‐of‐function with respect to PKC activity. However, we were interested in exploring whether regulatory domain fusions could also act in a dominant‐negative manner. Overexpression of PKCα‐CDH8 in cells suppressed endogenous PKC activity, both basally and following agonist stimulation. This suggests that PKC regulatory fusions are not only loss‐of‐function by loss of the catalytic domain, but also dominant negative by competition of the ligand‐sensing modules for binding the allosteric activator diacylglycerol. Taken together, our data demonstrate that both catalytic and regulatory domain PKC fusions are loss‐of‐function, revealing another mechanism by which PKC activity is lost in cancer and supporting a tumor suppressive role for PKC. Support or Funding Information This work was supported by NIH R35 GM122523 (ACN). ANV, TRB, and CEA were supported by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology (T32 GM007752), and CEA was supported by an NSF Graduate Research Fellowship (DGE1144086).

Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disease caused by germline mutations in the diacylglycerol (DG)/Ca2+‐regulated protein kinase C gamma (PKCγ), leading to Purkinje cell degeneration and progressive cerebellar dysfunction. Curiously, the majority of the approximately 50 missense mutations identified in PKCγ cluster to the DG‐sensing C1A and C1B domains. Here, we use a genetically‐encoded FRET‐based C Kinase Activity Reporter (CKAR) to show that ataxia‐associated PKCγ mutants have higher basal activity in cells, and thus are less autoinhibited, than wild‐type enzyme. However, whereas reduced autoinhibition generally renders PKC sensitive to degradation, we show that mutations in the C1B domain allow translocation to membranes but protect PKCγ from phorbol ester‐induced down‐regulation. Indeed, deletion of the C1B domain prevents PKCγ down‐regulation with phorbol esters, potent ligands for the C1 domains. Strikingly, the degree of impaired autoinhibition correlates inversely with age of disease onset. Patients with the most severe mutation we examined (V138E) present with symptoms as young children, whereas symptoms in patients with the least severe mutation examined (D115Y) manifested in their 40s. To understand the structural basis of mutations outside the C1 domains, we generated a model of PKCγ using homology modeling and molecular docking. Mutations outside the C1 domains occur in regions also predicted to disrupt autoinhibition, including the pseudosubstrate, a predicted interface between the kinase and C1B domains, and the C‐terminal tail of PKCγ. Taken together, our data support a model in which SCA14 mutations enhance PKCγ activity without compromising stability. Furthermore, because many of the genetic causes of the 40+ types of SCA alter Ca2+ homeostasis, deregulated PKCγ activity may be a common cause for the disease. This raises the possibility that inhibition of PKCγ will be a potentially viable therapeutic target for SCA. Support or Funding Information NIH T32 GM007752

It is well known that cells, especially cancer cells, have an ability of evasion of apoptosis by cellular stress like nutrient starvation. And the balance between apoptosis signal and apoptosis‐resistant signal will determine the fate of cells, dead or alive. Here we found new cell signaling system that plays a role in applying the brakes to cell death that in case cancer cells avoid apoptosis by cellular stress like starvation using newly developed biosensor and in silico docking simulation technique. The new cell signaling system is that second messenger, sphingosine 1‐phosphate (S1P), directly activates a key cell signaling protein, atypical protein kinase C (aPKC). First, we found that the inhibition of aPKC induces apoptosis of cancer cell lines. Next, for making clear the molecular mechanism of the aPKC‐induced apoptosis resistance, we generated a genetically encoded reporter with the same modular structure as the original C kinase activity reporter (CKAR) but with a unique substrate sequence that allows specific visualization of atypical PKC activity in cells. Using the atypical PKC‐specific CKAR (aCKAR) we found that intracellular S1P induces the activation of atypical PKC in an S1P receptor‐independent manner. Biochemical studies revealed that S1P directly binds to the kinase domain of atypical PKC isozymes, relieving autoinhibitory constraints to activate the enzyme. In silico docking studies were used to identify potential binding sites for aPKC, one of which was validated by biochemical and aCKAR imaging techniques. Now we got new insights about the player of evasion of apoptosis in cancer at the molecular level, and it has potential for development of new molecular‐targeted agents to release brakes against cell death. Support or Funding Information This study was supported by NIH to A.C.N., JSPS KAKENHI, Kobe University Grant for Japan‐US Collaboration, Nakatani Foundation Grant for Technology Development Research to T.K., NIH, HHMI, NBCR, NSF to J.A.M., JSPS KAKENHI to S.N. and T.O. A.D.C. was supported in part by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology.

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