Reciprocal Immunoprecipitation Research Articles

Abstract 3057: The TARZN complex binds to de novo enhancer mutations and promotes oncogenic expression in T-ALL

Abstract TAL1 is overexpressed in 40-60% of T-cell acute lymphoblastic leukaemia (T-ALL) cases and forms an oncogenic core regulatory circuit (CRC) with other transcription factors such as LMO1, LMO2 and GATA3. In 5% of T-ALL cases an insertion of a consensus GT dinucleotide is observed upstream of the TAL1 gene, termed the “Mutation of TAL1 enhancer” (MuTE), driving TAL1 overexpression. Using an in vitro reconstitution DNA pull-down assay combined with quantitative mass spectrometry, we identified nine candidate proteins that preferentially bound to the MuTE sequence compared to the WT sequence. Among the candidates, we demonstrate by reciprocal immunoprecipitation experiments coupled to quantitative mass spectrometry that the RNA methyltransferase TARBP1 and the zinc finger proteins ZBTB2, ZBTB25 and ZNF639 formed a complex that we term TARZN. Interestingly, the TARZN complex also bound to de novo super enhancer sites upstream of the LMO1 and LMO2 genes in T-ALL cells, indicating a putative common mechanism between these different non-coding driver mutations. Furthermore, knock-down of all TARZN members resulted in lower TAL1 protein expression in MuTE-positive but not in MuTE-negative T-ALL cells. Overall, these data suggest that the TARZN complex cooperatively promotes oncogenic expression in T-ALL. Citation Format: Nurkaiyisah Zaal Anuar, Chai Yeen Goh, Boon Haow Chua, Shi Hao Tan, Joana R. Costa, Marc R. Mansour, Takaomi Sanda, Dennis Kappei. The TARZN complex binds to de novo enhancer mutations and promotes oncogenic expression in T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3057.

Tetramer Formation of BCL11A Is Required for Both Protein Stability and Fetal Hemoglobin Silencing

The zinc-finger (ZnF) repressor protein BCL11A regulates the switch from fetal (HbF, α 2γ 2) to adult hemoglobin (HbA, α 2β 2) through direct binding at the γ-globin promoter. Clinical trials of gene-based reduction of BCL11A with ex vivo modified hematopoietic stem cells (HSCs) have yielded transformative outcomes in patients with sickle cell disease and β-thalassemia, establishing BCL11A as a therapeutic target. Unless in vivo methods for delivery of gene-based therapies become efficient enough to modify a substantial fraction of host HSCs, small molecule drugs will be required to reduce overall disease burden. With the goal of targeting BCL11A with small molecules, we have characterized its native state. The protein is largely unstructured, except for 7 ZnFs, numbered 0-6. Previously we showed that BCL11A protein appears stable in cells with a T 1/2 of ~ 24 hrs. Here we report the unexpected finding that the protein assembles and functions as a tetramer. Recombinant BCL11A behaves as a tetramer in biophysical assays. Gene editing of the endogenous BCL11A locus in CD34-derived, immortal HUDEP2 cells has been employed to correlate protein structure and expression with consequences of a given modification. We found that BCL11A lacking ZnF0 is unstable, leading to protein loss and derepression of γ-globin. Recombinant ZnF0 self-assembles into a stable tetramer, whose X-ray structure reveals a hydrophobic interface between subunits. Mutation of residues in the hydrophobic core disrupts tetramerization and markedly reduces steady-state protein in cells. Reciprocal immunoprecipitation in cells reveals that ZnF0 is both necessary and sufficient to mediate BCL11A mutlimer assembly. Collectively, these results indicate that BCL11A tetramerization is required for protein stability. In cells expressing BCL11A lacking ZnF0, the steady-state protein level is partially restored by addition of proteasome inhibitors, suggesting that mutant protein is actively degraded by the ubiquitin-proteasome system. In contrast to findings with ZnF0, discrete removal of ZnF1 within the context of full-length BCL11A results in a marked increase in steady-state protein in cells. In vitro purified ZnF1 forms a dimer and only dimeric ZnF1 can be stably expressed in cells. These findings re consistent with a model in which assembly of the BCL11A tetramer is required to mask a degron in ZnF1 that becomes obscured on its dimerization. Masking of a degron in ZnF1 predicts that discrete removal of both ZnF0 and ZnF1 within full-length BCL11A will lead to production of a monomer. Indeed, HUDEP2 cells expressing BCL11A lacking both ZnFs exhibit an elevated level of steady-state, monomeric protein, consistent with the degron masking model. However, despite an elevated level, cells expressing monomeric BCL11A are defective in HbF silencing, indicating that the tetramer structure is critical for γ-globin repression. Our findings provide novel insights into BCL11A action and suggest new strategies for therapeutic targeting. Combining structural, biochemical, and cellular approaches, we have shown that ZnF-mediated tetramer formation shields BCL11A from proteolytic degradation, and that the tetramer state itself is required for effective γ-globin repression. Of relevance to BCL11A as a therapeutic target, the tetramer of ZnF0 constitutes a structured, stable domain in an otherwise largely unstructured protein, and thereby offers possibilities for small molecule targeting. The multimer state of BCL11A is a vulnerability for HbF repression.

Identification and characterization of ARID1A-interacting proteins in renal tubular cells and their molecular regulation of angiogenesis

BackgroundDefects and deficiency of AT-rich interactive domain-containing protein 1A (ARID1A) encoded by a tumor suppressor gene ARID1A have recently been suggested to get involved in angiogenesis, a crucial process in carcinogenesis. However, molecular mechanisms of ARID1A deficiency to induce angiogenesis in kidney cancer remain underinvestigated.MethodsWe performed large-scale identification of ARID1A protein interactors in renal tubular epithelial cells (RTECs) using immunoprecipitation (IP) followed by nanoLC-ESI-LTQ-Orbitrap tandem mass spectrometry (MS/MS). Their roles in angiogenesis were investigated using various assays.ResultsA total of 74 ARID1A-interacting proteins were identified. Protein–protein interactions analysis revealed that these identified proteins interacted directly or indirectly with ARID1A. Among them, the direct interaction between ARID1A and β-actin was validated by IP and reciprocal IP followed by Western blotting. Small interfering RNA (siRNA) was used for single and double knockdowns of ARID1A and ACTB. Semi-quantitative RT-PCR demonstrated that deficiency of ARID1A, but not ACTB, significantly affected expression of angiogenesis-related genes in RTECs (VEGF and FGF2 were increased, whereas PDGF and EGF were decreased). However, the knockdowns did not affect TGFB1 and FGF1 levels. The quantitative mRNA expression data of VEGF and TGFB1 were consistent with the secreted levels of their protein products as measured by ELISA. Only secreted products derived from ARID1A-deficient RTECs significantly increased endothelial cells (ECs) migration and tube formation. Some of the other carcinogenic features could also be confirmed in the ARID1A-deficient RTECs, including increased cell migration and chemoresistance. Double knockdowns of both ARID1A and ACTB did not enhance the effects of single ARID1A knockdown in all assays.ConclusionsWe report herein a large dataset of the ARID1A-interacting proteins in RTECs using an IP-MS/MS approach and confirm the direct interaction between ARID1A and β-actin. However, the role of ARID1A deficiency in angiogenesis is independent of β-actin.

Abstract 2332: VSTM2A modulation of immune response in colorectal cancer through abrogating PD-L1 and PD-1 interaction

Abstract Background: Checkpoint immunotherapy using humanized antibody is a new therapeutic strategy against advanced colorectal cancer (CRC). Nonetheless, it shows low tumor penetration due to the large molecule size. Therefore, small immune regulatory molecules are actively sought. Our previous study showed that a small secretory protein VSTM2A suppressed Wnt signalling pathway through inducing LRP6 lysosome dependent degradation. However, its role in immune modulation is largely unknown. Methods: Clinicopathological features were analyzed using CRC patient cohort. Functional biology of VSTM2A was investigated using T cell-antigen presenting cell (APC) co-culture models and knockout mice model. Liquid chromatography with tandem mass spectrometry (LC-MS) was carried out for mechanistic investigation. Results: Gene ontology analysis of colon RNA-seq dataset from TCGA/GTEx revealed significant enrichment of adaptive immune response and lymphocyte activation with high VSTM2A expression. CRC patients with inferior VSTM2A expressions were associated with lower immunoscores in both early and late stages of CRC. In addition, our immunohistochemical examinations of 208 in-house CRC cases further pinpoints to a considerable fraction of VSTM2A-negative cases showing avoidance of spontaneous CD8a+ cells infiltration (Pearson's chi-squared test P < 0.001, φ = 0.24) and worse clinical outcome. LC-MS assay followed by reciprocal immunoprecipitation revealed that VSTM2A interacted with the IgV motif of immune checkpoint protein programmed cell death 1 ligand 1 (PD-L1). We further found that VSTM2A recognized PD-L1 with a dissociation constant (Kd) of 2.6 nM. Recombinant VSTM2A dose dependently suppressed the interaction of PD-L1 and PD-1 with IC50 of 4.51 nM, indicating VSTM2A is a potential intrinsic PD-L1 antagonist. Moreover, our in-vitro mechanistic studies revealed that VSTM2A protein could block the interaction of PD-1/PD-L1, and induced NFAT-RE luciferase activity dose-dependently using a bioluminescent cell-based PD-1/PD-L1 blockade bioassay. VSTM2A could dramatically activate the interleukin-2 production from DO11.10 T cell in the presence non-T cell spherocyte isolated from BALB/c mice and OVA323-339 peptide. Furthermore, our in-vivo study revealed that VSTM2A triggered a distinct increase in CD45+CD3+ T cells infiltration in the syngeneic MC38 mouse model. Vstm2a+/- knockout mice challenged with azoxymethane and dextran sodium sulfate showed significantly high CRC incidence and tumor burden in comparison to wild-type littermates. Tumor from Vstm2a+/- mice showed significantly less CD8+ T cell infiltration. Conclusions: our study demonstrate the tumor extrinsic role of VSTM2A in modulation of microenvironment through abrogating PD-L1/PD-1 interaction. Silencing of VSTM2A in CRC could suppress T cell activation and infiltration in the tumor sites. Citation Format: Yujuan Dong, Yunfei Zhou, Nathalie Wong, Jun Yu, Siu Man Simon Ng. VSTM2A modulation of immune response in colorectal cancer through abrogating PD-L1 and PD-1 interaction [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 2332.

Apically-located P4-ATPase1-Lem1 complex internalizes phosphatidylserine and regulates motility-dependent invasion and egress in Toxoplasma gondii

The membrane asymmetry regulated by P4-ATPases is crucial for the functioning of eukaryotic cells. The underlying spatial translocation or flipping of specific lipids is usually assured by respective P4-ATPases coupled to conforming non-catalytic subunits. Our previous work has identified five P4-ATPases (TgP4-ATPase1–5) and three non-catalytic partner proteins (TgLem1–3) in the intracellular protozoan pathogen Toxoplasma gondii. However, their flipping activity, physiological relevance and functional coupling remain unknown. Herein, we demonstrate that TgP4-ATPase1 and TgLem1 work together to translocate phosphatidylserine (PtdSer) during the lytic cycle of T. gondii. Both proteins localize in the plasma membrane at the invasive (apical) end of its acutely-infectious tachyzoite stage. The genetic knockout of P4-ATPase1 and conditional depletion of Lem1 in tachyzoites severely disrupt the asexual reproduction and translocation of PtdSer across the plasma membrane. Moreover, the phenotypic analysis of individual mutants revealed a requirement of lipid flipping for the motility, egress and invasion of tachyzoites. Not least, the proximity-dependent biotinylation and reciprocal immunoprecipitation assays demonstrated the physical interaction of P4-ATPase1 and Lem1. Our findings disclose the mechanism and significance of PtdSer flipping during the lytic cycle and identify the P4-ATPase1-Lem1 heterocomplex as a potential drug target in T. gondii.

USP10 deubiquitinates Tau, mediating its aggregation

Normal Tau promotes the assembly and stabilization of microtubules, thus, maintaining axon transport. In Alzheimer’s disease (AD), Tau aggregation causes it to lose these above-mentioned functions. However, the molecular mechanism leading to Tau aggregation in AD remains ambiguous. Here, we report that USP10, one of the important deubiquitinases (DUBs), is involved in Tau aggregation. We found that USP10 is upregulated in postmortem human AD and APP/PS1 mice brains, but not in P301S mice brains. Moreover, in primary neuronal cultures, Aβ42 induces a dose-dependent USP10 upregulation, an increase in the levels of both total and phosphorylated Tau, as well as a markedly elevated Tau binding with USP10, that is accompanied by a significantly decreased Tau ubiquitination. In addition, overexpression of USP10 directly causes an increase in the levels of total and phosphorylated Tau, induces Tau aggregation, and delays in Tau degradation. Results from mass spectrometry, reciprocal immunoprecipitation, and immunofluorescence assays strongly prove Tau’s interaction with USP10. This is further supported by the Tau307–326K and Tau341–378K peptides’ competitive inhibition of Tau binding with USP10, attenuating Tau hyperphosphorylation and Tau deubiquitination. Together, our data strongly indicate that USP10 plays a critical role in mediating Tau aggregation via downregulating its ubiquitination and thus slowing down Tau turnover. Inhibition of USP10-Tau interaction might be therapeutically useful in the management of AD and related tauopathies.

Abstract 866: Translational repression by a novel partner of human cytoplasmic poly(A) binding protein

Abstract In higher eukaryotes, post-transcriptional regulation of gene expression, which is accomplished by an ensemble of RNA-binding proteins (RBPs), is critical for cellular homeostasis. Unkempt (UNK) is an evolutionarily conserved Zinc Finger/RING domain RNA-binding protein that was originally identified as a developmental regulator in Drosophila. Recent studies suggest that UNK targets specific mRNAs through its two compact zinc finger clusters and regulates their translation. However, the underlying molecular mechanisms by which UNK represses translation remain unclear. To examine UNK interactions, we purified Flag-tagged UNK and subjected the preparations to Mass spectrometry (MS) and identified an enriched protein Cytoplasmic poly(A)-binding protein(PABPC1), a key component of the translation machinery that binds to the poly(A) tail of mRNAs to promote translation and mRNA turnover. GST Pull down assay and reciprocal immunoprecipitations in HEK293 cells further confirmed the strong interaction between UNK and PABPC1 and showed that the interaction is RNA-dependent. Notably, the structural analysis, along with mutational studies proved that the interaction is mediated by the C-terminal MLLE domain of PABPC1 and the C-terminal Domain of UNK both in vitro and in vivo. Further MS2 tethering assay indicated that UNK inhibits PABPC1-stimulated translation activity. Our studies identified Unkempt as a novel partner of PABPC1 that functions to represses PABPC1-stimulated translation. The findings provide novel insight into the molecular function of Unkempt and PABPC1-mediated translational machinery. Citation Format: Naren Li, Liang Hu, Qinfang Liu, Yulan Xiong, Jianzhong Yu. Translational repression by a novel partner of human cytoplasmic poly(A) binding protein [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 866.

Abstract LB038: Identification of androgen receptor co-chaperone interactors implicated in castration resistant prostate cancer

Abstract Background: Prostate cancer (PCa) is the most common non-cutaneous cancer afflicting men, both in the United States and worldwide. Aberrant molecular signaling mechanisms mediated by the Androgen Receptor (AR) are attributed as the main culprits for the initiation and progression of PCa, and research efforts have focused on developing strategies to directly target the AR. In an alternative approach, molecular chaperones that are critical for the maturation and signaling of the AR have emerged as potential therapeutic targets. Our lab has demonstrated the potential of inhibitors that target AR cochaperones, FKBP51 and FKBP52, in PCa cells. Previously, FKBP51 and FKBP52 have been shown to play a role in cell proliferation and survival, steroid hormone receptor maturation, and other mechanisms highly implicated in prostate carcinogenesis. Therefore, it is imperative to understand the protein-protein interactions that regulate FKBP51 and FKBP52-mediated AR function in PCa. We hypothesize that unknown interactors cooperate with FKBP51 and FKBP52 to promote PCa progression via the AR signaling pathways. Here, we performed a series of experiments aimed to identify and characterize auxiliary proteins influencing FKBP51- and FKBP52-mediated AR activity. Ultimately, our goal is to define novel protein interactions involved in AR-mediated PCa progression for therapeutic intervention. Methods: We implemented tandem affinity purification followed by mass spectrometry analysis in 22RV1 PCa cells using FKBP51 and FKBP52 as bait. The spectral counts of proteins identified were then used to analyze protein-protein interactions through the IDEP workflow software. Gene Ontology (GO) term enrichment analysis was used to generate a STRING interaction network. Protein interactions of interest were then validated via reciprocal immunoprecipitations. Results: This interactome analysis resulted in 353 IDs for FKBP51 and FKBP52 interactors. Heatmaps representing top 100 interactors were generated for both bait proteins. Members of the peroxiredoxin family, implicated in development of PCa and resistance to treatment, are among the top candidates of interest resulting from this interactome. Conclusion: This analysis aims to provide important insights into the androgen signaling milieu, which can be applied in the development of novel treatments targeting AR interacting partners in PCa progression. This study contributes to the growing list of functionally diverse proteins that help modulate AR transcriptional activity, and supports the idea that AR signaling is dynamic with numerous molecules involved in the aberrant activation of AR in prostate-tumor cells Citation Format: Olga B. Soto, Abhijeet R. Patil, Sourav Roy, Marc B. Cox. Identification of androgen receptor co-chaperone interactors implicated in castration resistant prostate cancer [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 LB038.

Protein interactome and cell-type expression analyses reveal that cytoplasmic FMR1-interacting protein 1 (CYFIP1), but not CYFIP2, associates with astrocytic focal adhesion.

The two members of the cytoplasmic FMR1-interacting protein family, CYFIP1 and CYFIP2, are evolutionarily conserved multifunctional proteins whose defects are associated with distinct types of brain disorders. Even with high sequence homology between CYFIP1 and CYFIP2, several lines of evidence indicate their different functions in the brain; however, the underlying mechanisms remain largely unknown. Here, we performed reciprocal immunoprecipitation experiments using CYFIP1-2× Myc and CYFIP2-3× Flag knock-in mice and found that CYFIP1 and CYFIP2 are not significantly co-immunoprecipitated with each other in the knock-in brains compared with negative control wild-type (WT) brains. Moreover, CYFIP1 and CYFIP2 showed different size distributions by size-exclusion chromatography of WT mouse brains. Specifically, mass spectrometry-based analysis of CYFIP1-2× Myc knock-in brains identified 131 proteins in the CYFIP1 interactome. Comparison of the CYFIP1 interactome with the previously identified brain region- and age-matched CYFIP2 interactome, consisting of 140 proteins, revealed only eight common proteins. Investigations using single-cell RNA-sequencing databases suggested non-neuronal cell- and neuron-enriched expression of Cyfip1 and Cyfip2, respectively. At the protein level, CYFIP1 was detected in both neurons and astrocytes, while CYFIP2 was detected only in neurons, suggesting the predominant expression of CYFIP1 in astrocytes. Bioinformatic characterization of the CYFIP1 interactome, and co-expression analysis of Cyfip1 with astrocytic genes, commonly linked CYFIP1 with focal adhesion proteins. Immunocytochemical analysis and proximity ligation assay suggested partial co-localization of CYFIP1 and focal adhesion proteins in cultured astrocytes. Together, these results suggest a CYFIP1-specific association with astrocytic focal adhesion, which may contribute to the different brain functions and dysfunctions of CYFIP1 and CYFIP2. Cover Image for this issue: https://doi.org/10.1111/jnc.15410.

UPF1 promotes rapid degradation of m6A-containing RNAs

N6-methyladenosine (m6A) is the most prevalent internal modification in eukaryotic mRNAs and affects RNA processing and metabolism. When YTHDF2, an m6A-recognizing protein, binds to m6A, it facilitates the destabilization of m6A-containing RNAs (m6A RNAs). Here, we demonstrate that upstream frameshift 1 (UPF1), a key factor for nonsense-mediated mRNA decay, interacts with YTHDF2, thereby triggering rapid degradation of m6A RNAs. The UPF1-mediated m6A RNA degradation depends on a specific interaction between UPF1 and N-terminal residues 101-168 of YTHDF2, UPF1 ATPase/helicase activities, and UPF1 interaction with proline-rich nuclear receptor coactivator 2 (PNRC2), a decapping-promoting factor preferentially involved in nonsense-mediated mRNA decay. Furthermore, transcriptome-wide analyses show that YTHDF2-bound mRNAs that are not substrates for HRSP12-RNase P/MRP-mediated endoribonucleolytic cleavage are destabilized with a higher dependency on UPF1. Collectively, our data indicate dynamic and multilayered regulation of the stability of m6A RNAs and highlight the multifaceted role of UPF1 in mRNA decay.

RNAmetasome network for macromolecule biogenesis in human cells

RNA plays a central role in macromolecule biogenesis for various pathways, such as gene expression, ribosome biogenesis, and chromatin remodeling. However, RNA must be converted from its nascent to functional forms for that role. Here, we describe a large RNA metabolic network (RNAmetasome network) for macromolecule biogenesis in human cells. In HEK293T, the network consists of proteins responsible for gene expression, splicing, ribosome biogenesis, chromatin remodeling, and cell cycle. Reciprocal immunoprecipitations show that MKI67, GNL2, MDN1, and ELMSAN1 are core proteins of the network, and knockdown of either MKI67 or GNL2 affects the state of the other protein, MDN1, and some other network members. Furthermore, GNL2 knockdown retards cell proliferation. Several proteins of the RNAmetasome network are diminished in Hela.cl1, and this diminishment is associated with low expression of MDN1 and elevated MKI67 degradation. These results together suggest that the RNAmetasome network is present in human cells and associated with proliferation, and that MKI67, GNL2, and MDN1 play an important role in organizing the RNAmetasome network.

Characterisation of complexes formed by parasite proteins exported into the host cell compartment of Plasmodium falciparum infected red blood cells.

During its intraerythrocytic life cycle, the human malaria parasite Plasmodium falciparum supplements its nutritional requirements by scavenging substrates from the plasma through the new permeability pathways (NPPs) installed in the red blood cell (RBC) membrane. Parasite proteins of the RhopH complex: CLAG3, RhopH2, RhopH3, have been implicated in NPP activity. Here, we studied 13 exported proteins previously hypothesised to interact with RhopH2, to study their potential contribution to the function of NPPs. NPP activity assays revealed that the 13 proteins do not appear to be individually important for NPP function, as conditional knockdown of these proteins had no effect on sorbitol uptake. Intriguingly, reciprocal immunoprecipitation assays showed that five of the 13 proteins interact with all members of the RhopH complex, with PF3D7_1401200 showing the strongest association. Mass spectrometry‐based proteomics further identified new protein complexes; a cytoskeletal complex and a Maurer's clefts/J‐dot complex, which overall helps clarify protein–protein interactions within the infected RBC (iRBC) and is suggestive of the potential trafficking route of the RhopH complex itself to the RBC membrane.

HOMER3 facilitates growth factor-mediated \u03b2-Catenin tyrosine phosphorylation and activation to promote metastasis in triple negative breast cancer

BackgroundHOMER family scaffolding proteins (HOMER1-3) play critical roles in the development and progression of human disease by regulating the assembly of signal transduction complexes in response to extrinsic stimuli. However, the role of HOMER protein in breast cancer remains unclear.MethodsHOMER3 expression was examined by immunohistochemistry in breast cancer patient specimens, and its significance in prognosis was assessed by Kaplan–Meier survival analysis. The effects of HOMER3 in growth factor-induced β-Catenin activation were analyzed by assays such as TOP/FOP flash reporter, tyrosine phosphorylation assay and reciprocal immunoprecipitation (IP) assay. Role of HOMER3 in breast cancer metastasis was determined by cell function assays and mice tumor models.ResultsHerein, we find that, among the three HOMER proteins, HOMER3 is selectively overexpressed in the most aggressive triple negative breast cancer (TNBC) subtype, and significantly correlates with earlier tumor metastasis and shorter patient survival. Mechanismly, HOMER3 interacts with both c-Src and β-Catenin, thus providing a scaffolding platform to facilitate c-Src-induced β-Catenin tyrosine phosphorylation under growth factor stimulation. HOMER3 promotes β-Catenin nuclear translocation and activation, and this axis is clinically relevant. HOMER3 promotes and is essential for EGF-induced aggressiveness and metastasis of TNBC cells both in vitro and in vivo.ConclusionThese findings identify a novel role of HOMER3 in the transduction of growth factor-mediated β-Catenin activation and suggest that HOMER3 might be a targetable vulnerability of TNBC.

Abstract S01-01: Therapeutic targeting of TMPRSS2 and ACE2 as a potential strategy to combat COVID-19

Abstract The novel SARS-CoV-2 infection responsible for the COVID-19 pandemic is expected to have an adverse effect on the progression of multiple cancers, including prostate cancer, due to the ensuing cytokine storm associated oncogenic signaling. A better understanding of the host cell factors and their regulators will help identify potential therapies to block SARS-CoV-2 infection at an early stage and thereby prevent cancer progression. Host cell infection by SARS-CoV-2 requires the binding of the viral spike S protein to ACE2 receptor and priming by the serine protease TMPRSS2—encoded by a well-known androgen response gene and highly expressed in patients diagnosed with prostate cancer. Epidemiologic data showing increased severity and mortality of SARS-CoV-2 disease in men suggest a possible role for androgen in the transcriptional activation of ACE2 and TMPRSS2 in the lungs and other primary infection sites. Here, by performing in vivo castration in mice, RT-PCR, immunoblotting, Co-IP, and pseudovirus infection assays in multiple cell lines, we present evidence for the transcriptional regulation of TMPRSS2 and ACE2 by androgen, their endogenous interaction, as well as a novel combination of drugs in blocking viral infection. In adult male mice, castration led to a significant loss in the expression of ACE2 and TMPRSS2 at the transcript and protein levels in the lung, heart, and small intestine. Intriguingly, castrated mice displayed a substantial increase in ACE2 in the kidney, which could potentially be due to the low blood pressure resulting from androgen deprivation. Endogenous TMPRSS2 and ACE2 were found to be physically interacting, as observed by reciprocal immunoprecipitation followed by immunoblotting. Importantly, along with full-length zymogen form, a prominent novel small isoform of TMPRSS2 was found to be associated with ACE2 in lung and prostate cells. In an overexpression system, camostat—a serine protease inhibitor specific to TMPRSS2—inhibited the cleavage of spike S, suggesting a direct role of this serine protease in priming the viral spike S protein, a prerequisite for an active infection. Furthermore, a combination of camostat, antiandrogen, and epigenetic drugs at sublethal concentrations blocked SARS-CoV-2 pseudovirus infection in multiple cell types. Together, our preclinical data provide a strong rationale for clinical evaluation of TMRPSS2 inhibitors, antiandrogens, and epigenetic drug combinations along with antiviral drug remdesivir as early as clinically possible to prevent progression to pneumonia and multiorgan failure as a result of hyperinflammatory responses in COVID-19 patients. Citation Format: Qu Deng, Reyaz Ur Rasool, Ramakrishnan Natesan, Irfan A. Asangani. Therapeutic targeting of TMPRSS2 and ACE2 as a potential strategy to combat COVID-19 [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2020 Jul 20-22. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(18_Suppl):Abstract nr S01-01.

Cooperative roles of PAK1 and filamin A in regulation of vimentin assembly and cell extension formation

The formation of extensions in cell migration requires tightly coordinated reorganization of all three cytoskeletal polymers but the mechanisms by which intermediate filament networks interact with actin to generate extensions are not well-defined. We examined interactions of the actin binding protein filamin A (FLNA) with vimentin in extension formation by fibroblasts. Knockdown (KD) of vimentin in fibroblasts reduced the lengths of cell extensions by 50% (p < 0.001). After cell binding to fibronectin, there was a time-dependent increase of phosphorylation of serine 39, 56 and 72 in vimentin, which was associated with vimentin filament assembly. Of the FLNA-interacting kinases that could phosphorylate vimentin, we focused on PAK1, which we found by reciprocal immunoprecipitation associated with FLNA. Enzyme inhibitor studies and siRNA KD demonstrated that PAK1 was required for vimentin phosphorylation and formation of cell extensions. In sedimentation assays, vimentin was exclusively detected in the insoluble pellet fraction of cells expressing FLNA while in FLNA KD cells there was increased vimentin in the supernatants of FLN KD cells. Compared with wild type, FLNA KD cells showed loss of phosphorylation of serine 56 and 72 in vimentin and reduced numbers and lengths of cell extensions by >4-fold. We suggest that the association of PAK1 with FLNA enables vimentin phosphorylation and filament assembly, which are important in the development and stabilization of cell extensions during cell migration.

The mRNA encoding the JUND tumor suppressor detains nuclear RNA-binding proteins to assemble polysomes that are unaffected by mTOR

One long-standing knowledge gap is the role of nuclear proteins in mRNA translation. Nuclear RNA helicase A (DHX9/RHA) is necessary for the translation of the mRNAs of JUND (JunD proto-oncogene AP-1 transcription factor subunit) and HIV-1 genes, and nuclear cap-binding protein 1 (NCBP1)/CBP80 is a component of HIV-1 polysomes. The protein kinase mTOR activates canonical messenger ribonucleoproteins by post-translationally down-regulating the eIF4E inhibitory protein 4E-BP1. We posited here that NCBP1 and DHX9/RHA (RHA) support a translation pathway of JUND RNA that is independent of mTOR. We present evidence from reciprocal immunoprecipitation experiments indicating that NCBP1 and RHA both are components of messenger ribonucleoproteins in several cell types. Moreover, tandem affinity and RT-quantitative PCR results revealed that JUND mRNA is a component of a previously unknown ribonucleoprotein complex. Results from the tandem IP indicated that another component of the JUND-containing ribonucleoprotein complex is NCBP3, a recently identified ortholog of NCBP2/CBP20. We also found that NCBP1, NCBP3, and RHA, but not NCBP2, are components of JUND-containing polysomes. Mutational analysis uncovered two dsRNA-binding domains of RHA that are necessary to tether JUND-NCBP1/NCBP3 to polysomes. We also found that JUND translation is unaffected by inhibition of mTOR, unless RHA was down-regulated by siRNA. These findings uncover a noncanonical cap-binding complex consisting of NCBP1/NCBP3 and RHA substitutes for the eukaryotic translation initiation factors 4E and 4G and activates mTOR-independent translation of the mRNA encoding the tumor suppressor JUND.

Interaction of Plasmodium falciparum casein kinase 1 with components of host cell protein trafficking machinery.

A pool of Plasmodium falciparum casein kinase 1 (PfCK1) has been shown to localize to the host red blood cell (RBC) membrane and be secreted to the extracellular medium during trophozoite stage of development. We attempted to identify mechanisms for secretion of PfCK1 and its appearance on the RBC membrane. We found that two host proteins with established functions in membrane trafficking in higher eukaryotes, GTPase-activating protein and Vps9 domain-containing protein 1 (GAPVD1), and Sorting nexin 22, consistently co-purify with PfCK1, suggesting that the parasite utilizes trafficking pathways previously thought to be inactive in RBCs. Furthermore, reciprocal immunoprecipitation experiments with GAPVD1 identified parasite proteins suggestive of a protein recycling pathway hitherto only described in higher eukaryotes. Thus, we have identified components of a trafficking pathway involving parasite proteins that act in concert with host proteins, and which we hypothesize mediates trafficking of PfCK1 to the RBC during infection.

Protein tyrosine phosphatase SHP-1 modulates microtubule organization in later stages of mast cell activation

Abstract Antigen-mediated activation of mast cells initiates signaling events leading to degranulation and release of inflammatory mediators. Although rapid and transient microtubule reorganization during activation has been described, the molecular mechanisms that control their rearrangement are largely unknown. Important role in microtubule nucleation from centrosomes play γ-tubulin complexes. Here we report on the regulation of microtubule organization in bone marrow-derived mast cells (BMMCs) by Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 1 (SHP-1). Reciprocal immunoprecipitation experiments revealed that SHP-1 is present in complexes containing γ-tubulin complex proteins and protein tyrosine kinase Syk. Pull-down experiments with truncated forms of γ-tubulin and SHP-1 unveiled corresponding interaction domains. When compared to controls, activated BMMCs without SHP-1 had more protrusions containing microtubules, higher level of tyrosine phosphorylation, enhanced activity of Syk kinase, as well as increased expression of cytokines and prostaglandins. Intracellular calcium mobilization and degranulation also increased. Microtubule regrowth experiments in cells lacking SHP-1 revealed stimulation of microtubule nucleation, and phenotypic rescue experiments confirmed that SHP-1 represents a negative regulator of microtubule nucleation in BMMCs. Moreover, inhibition of the SHP-1 activity by inhibitors TPI-1 and NSC87877 also augmented microtubule nucleation. The regulation was due to changes of γ-tubulin accumulation in centrosomes. Our data suggest a novel SHP-1 dependent mechanism for the suppression of microtubule formation in later stages of mast cell activation.

Abstract P3-01-06: Decorin-mediated suppression of tumorigenesis and skin invasion in inflammatory breast cancer via inhibition of the E-cadherin/EGFR axis

Abstract Background: Inflammatory breast cancer (IBC) is a clinically distinct and highly aggressive form of primary breast cancer. Although considered rare, IBC accounts for 10% of breast cancer-related deaths owing to its rapid proliferation and strong propensity to metastasize. The molecular mechanisms underlying the aggressiveness and metastatic propensity of IBC remain elusive. Through transcriptome profiling we identified Decorin (DCN) as being significantly altered in metastatic IBC cell sublines. DCN is a secreted, small leucine-rich proteoglycan known to function as a tumor/metastasis suppressor by inhibiting several signaling pathways including EGFR, TGFβ, and c-MET. However, whether or how DCN regulates IBC tumorigenesis or metastasis is unknown. The aim of this study was to investigate the function and mechanism of DCN in IBC tumorigenesis and metastasis. Methods: Three IBC cell lines [ER-/HER2+ (MDA-IBC3, SUM190) and ER-/HER2- (SUM149)] were used. DCN gene expression in clinical samples was analyzed from publically available datasets and the IBC Consortium dataset. DCN was stably expressed in IBC cell lines by using lentiviral vectors. For in vivo studies, DCN-overexpressing stable cell lines were injected into cleared mammary fat pads of SCID/Beige mice and tumor growth was monitored via caliper measurements. Tumor-skin involvement was assessed visually during primary tumor growth and tumor excision. Reverse phase protein array analysis was used for proteomic profiling. Protein-protein interactions were analyzed by reciprocal immunoprecipitation of exogenous or endogenous proteins. Results: DCN expression was significantly lower in breast cancer samples than in normal breast (p&amp;lt;0.0001), in more aggressive breast cancer subtypes (p&amp;lt;0.0001), and in metastatic tumors relative to primary tumors (p&amp;lt;0.0001). High DCN expression correlated with improved overall survival (p&amp;lt;0.0001) and relapse-free survival (p=0.0003). In vitro, DCN overexpression in IBC cell lines inhibited colony formation (MDA-IBC3, p=0.0115; SUM149, p=0.0068), migration (SUM149, p=0.0165), invasion (SUM149, p=0.0159), and primary and secondary mammosphere formation (primary: MDA-IBC3, p=0.0209; SUM149, p=0.0232; secondary: MDA-IBC3, p=0.01; SUM149, p=0.0031). In vivo, DCN overexpression in MDA-IBC3 cells inhibited primary tumor growth (p=0.0092) and reduced skin invasion (89.9% DCN control vs 33.3% DCN-overexpressed, p=0.017, Chi-square test). Our proteomics data in DCN-overexpressing SUM149 and MDA-IBC3 cells showed downregulated EGFR and E-cadherin protein levels. Mechanistically, DCN reduced expression of E-cadherin and EGFR and reduced phosphorylation of EGFR in IBC cells. Exogenous and endogenous co-immunoprecipitation experiments showed direct physical binding between DCN and E-cadherin proteins in all IBC cell lines. Moreover, overexpression of DCN downregulated E-cadherin via protein instability rather than decreased E-cadherin mRNA expression. E-cadherin knockdown in IBC cells decreased, whereas its overexpression increased, activation of EGFR signaling without affecting DCN expression. Finally, restoring E-cadherin in DCN-overexpressing IBC cell lines rescued the inhibitory effect of DCN on EGFR signaling. Conclusions: We found that DCN inhibited tumorigenesis and skin invasion in IBC via its direct interaction with and stabilization of E-cadherin and its suppression of EGFR signaling. Our findings provide new insights and a novel mechanism for IBC pathobiology that may be therapeutically targetable. Future studies will determine the role of DCN in IBC metastasis and the detailed mechanism of DCN-mediated suppression of tumorigenesis and metastasis in IBC. Citation Format: Xiaoding Hu, Emilly Schlee Villodre, Richard Larson, Omar M. Rahal, Xiaoping Wang, Savitri Krishnamurthy, Debu Tripathy, Naoto T Ueno, Wendy A Woodward, Bisrat Godefay Debeb. Decorin-mediated suppression of tumorigenesis and skin invasion in inflammatory breast cancer via inhibition of the E-cadherin/EGFR axis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-06.

IGF2BP2 regulates DANCR by serving as an N6-methyladenosine reader

The major function of Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) is to regulate cell metabolism. However, emerging evidence indicates that IGF2BP2 plays a role in cancer, but the underlying mechanism is largely unknown. Here we showed that upregulation of IGF2BP2 is associated with poor outcomes of pancreatic cancer patients and suppression of IGF2BP2 inhibits cell proliferation. We further showed that IGF2BP2 regulates lncRNA DANCR. Ectopic expression IGF2BP2 enhances, whereas knockdown (KD) or knockout (KO) of IGF2BP2 suppresses DANCR expression. Moreover, in vivo RNA precipitation and reciprocal RNA immunoprecipitation revealed that IGF2BP2 interacts with DANCR. DANCR promotes cell proliferation and stemness-like properties. Experiments with xenograft models revealed that while ectopic expression of DANCR promotes, DANCR KO suppresses tumor growth. Mechanistically, DANCR is modified at N6-methyladenosine (m6A) and mutagenesis assay identified that adenosine at 664 of DANCR is critical to the interaction between IGF2BP2 and DANCR where IGF2BP2 serves a reader for m6A modified DANCR and stabilizes DANCR RNA. Together, these results suggest that DANCR is a novel target for IGF2BP2 through m6A modification, and IGF2BP2 and DANCR work together to promote cancer stemness-like properties and pancreatic cancer pathogenesis.