Loss of a 7q gene, CUX1, disrupts epigenetically driven DNA repair and drives therapy-related myeloid neoplasms.

Therapy-related myeloid neoplasms (t-MNs) are high-risk late effects with poorly understood pathogenesis in cancer survivors. It has been postulated that, in some cases, hematopoietic stem and progenitor cells (HSPCs) harboring mutations are selected for by cytotoxic exposures and transform. Here, we evaluate this model in the context of deficiency of CUX1, a transcription factor encoded on chromosome 7q and deleted in half of t-MN cases. We report that CUX1 has a critical early role in the DNA repair process in HSPCs. Mechanistically, CUX1 recruits the histone methyltransferase EHMT2 to DNA breaks to promote downstream H3K9 and H3K27 methylation, phosphorylated ATM retention, subsequent γH2AX focus formation and propagation, and, ultimately, 53BP1 recruitment. Despite significant unrepaired DNA damage sustained in CUX1-deficient murine HSPCs after cytotoxic exposures, they continue to proliferate and expand, mimicking clonal hematopoiesis in patients postchemotherapy. As a consequence, preexisting CUX1 deficiency predisposes mice to highly penetrant and rapidly fatal therapy-related erythroleukemias. These findings establish the importance of epigenetic regulation of HSPC DNA repair and position CUX1 as a gatekeeper in myeloid transformation.

Gene dosage effect of CUX1 in a murine model disrupts HSC homeostasis and controls the severity and mortality of MDS.

Monosomy 7 (-7) and del(7q) are high-risk cytogenetic abnormalities common in myeloid malignancies. We previously reported that CUX1, a homeodomain-containing transcription factor encoded on 7q22, is frequently inactivated in myeloid neoplasms, and CUX1 myeloid tumor suppressor activity is conserved from humans to Drosophila. CUX1-inactivating mutations are recurrent in clonal hematopoiesis of indeterminate potential as well as myeloid malignancies, in which they independently carry a poor prognosis. To determine the role for CUX1 in hematopoiesis, we generated 2 short hairpin RNA-based mouse models with ∼54% (Cux1mid) or ∼12% (Cux1low) residual CUX1 protein. Cux1mid mice develop myelodysplastic syndrome (MDS) with anemia and trilineage dysplasia, whereas CUX1low mice developed MDS/myeloproliferative neoplasms and anemia. In diseased mice, restoration of CUX1 expression was sufficient to reverse the disease. CUX1 knockdown bone marrow transplant recipients exhibited a transient hematopoietic expansion, followed by a reduction of hematopoietic stem cells (HSCs), and fatal bone marrow failure, in a dose-dependent manner. RNA-sequencing after CUX1 knockdown in human CD34+ cells identified a -7/del(7q) MDS gene signature and altered differentiation, proliferative, and phosphatidylinositol 3-kinase (PI3K) signaling pathways. In functional assays, CUX1 maintained HSC quiescence and repressed proliferation. These homeostatic changes occurred in parallel with decreased expression of the PI3K inhibitor, Pik3ip1, and elevated PI3K/AKT signaling upon CUX1 knockdown. Our data support a model wherein CUX1 knockdown promotes PI3K signaling, drives HSC exit from quiescence and proliferation, and results in HSC exhaustion. Our results also demonstrate that reduction of a single 7q gene, Cux1, is sufficient to cause MDS in mice.

The haploinsufficient tumor suppressor, CUX1, acts as an analog transcriptional regulator that controls target genes through distal enhancers that loop to target promoters.

One third of tumor suppressors are haploinsufficient transcriptional regulators, yet it remains unknown how a 50% reduction of a transcription factor is translated at the cis-regulatory level into a malignant transcriptional program. We studied CUX1, a haploinsufficient transcription factor that is recurrently mutated in hematopoietic and solid tumors. We determined CUX1 DNA-binding and target gene regulation in the wildtype and haploinsufficient states. CUX1 binds with transcriptional activators and cohesin at distal enhancers across three different human cell types. Haploinsufficiency of CUX1 altered the expression of a large number of genes, including cell cycle regulators, with concomitant increased cellular proliferation. Surprisingly, CUX1 occupancy decreased genome-wide in the haploinsufficient state, and binding site affinity did not correlate with differential gene expression. Instead, differentially expressed genes had multiple, low-affinity CUX1 binding sites, features of analog gene regulation. A machine-learning algorithm determined that chromatin accessibility, enhancer activity, and distance to the transcription start site are features of dose-sensitive CUX1 transcriptional regulation. Moreover, CUX1 is enriched at sites of DNA looping, as determined by Hi-C analysis, and these loops connect CUX1 to the promoters of regulated genes. We propose an analog model for haploinsufficient transcriptional deregulation mediated by higher order genome architecture.

Inhibition of sphingosine kinase 2 downregulates the expression of c-Myc and Mcl-1 and induces apoptosis in multiple myeloma.

Sphingolipid metabolism is being increasingly recognized as a key pathway in regulating cancer cell survival and proliferation. However, very little is known about its role in multiple myeloma (MM). We investigated the potential of targeting sphingosine kinase 2 (SK2) for the treatment of MM. We found that SK2 was overexpressed in MM cell lines and in primary human bone marrow (BM) CD1381 myeloma cells. Inhibition of SK2 by SK2- specific short hairpin RNA or ABC294640 (a SK2 specific inhibitor) effectively inhibited myeloma cell proliferation and induced caspase 3­mediated apoptosis. ABC294640 inhibited primary human CD1381 myeloma cells with the same efficacy as with MM cell lines. ABC294640 effectively induced apoptosis of myeloma cells, even in the presence of BM stromal cells. Furthermore, we found that ABC294640 downregulated the expression of pS6 and directed c-Myc and myeloid cell leukemia 1 (Mcl-1) for proteasome degradation. In addition, ABC294640 increased Noxa gene transcription and protein expression. ABC294640, per se, did not affect the expression of B-cell lymphoma 2 (Bcl-2), but acted synergistically with ABT-737 (a Bcl-2 inhibitor) in inducing myeloma cell death. ABC294640 suppressed myeloma tumor growth in vivo in mouse myeloma xenograft models. Our data demonstrated that SK2 provides a novel therapeutic target for the treatment of MM.This trial was registered at www.clinicaltrials.gov as #NCT01410981.

Pim1 serine/threonine kinase regulates the number and functions of murine hematopoietic stem cells.

The genes and pathways that govern the functions and expansion of hematopoietic stem cells (HSC) are not completely understood. In this study, we investigated the roles of serine/threonine Pim kinases in hematopoiesis in mice. We generated PIM1 transgenic mice (Pim1-Tx) overexpressing human PIM1 driven by vav hematopoietic promoter/regulatory elements. Compared to wild-type littermates, Pim1-Tx mice showed enhanced hematopoiesis as demonstrated by increased numbers of Lin(-) Sca-1 (+) c-Kit (+) (LSK) hematopoietic stem/progenitor cells and cobblestone area forming cells, higher BrdU incorporation in long-term HSC population, and a better ability to reconstitute lethally irradiated mice. We then extended our study using Pim1(-/-), Pim2(-/-), Pim3(-/-) single knockout (KO) mice. HSCs from Pim1(-/-) KO mice showed impaired long-term hematopoietic repopulating capacity in secondary and competitive transplantations. Interestingly, these defects were not observed in HSCs from Pim2(-/-) or Pim3(-/-) KO mice. Limiting dilution competitive transplantation assay estimated that the frequency of LSKCD34(-) HSCs was reduced by approximately 28-fold in Pim1(-/-) KO mice compared to wild-type littermates. Mechanistic studies demonstrated an important role of Pim1 kinase in regulating HSC cell proliferation and survival. Finally, our polymerase chain reaction (PCR) array and confirmatory real-time PCR (RT-PCR) studies identified several genes including Lef-1, Pax5, and Gata1 in HSCs that were affected by Pim1 deletion. Our data provide the first direct evidence for the important role of Pim1 kinase in the regulation of HSCs. Our study also dissects out the relative role of individual Pim kinase in HSC functions and regulation.

Oncogenic RAS promotes leukemic transformation of CUX1-deficient cells.

-7/del(7q) is prevalent across subtypes of myeloid neoplasms. CUX1, located on 7q22, encodes a homeodomain-containing transcription factor, and, like -7/del(7q), CUX1 inactivating mutations independently carry a poor prognosis. As with loss of 7q, CUX1 mutations often occur early in disease pathogenesis. We reported that CUX1 deficiency causes myelodysplastic syndrome in mice but was insufficient to drive acute myeloid leukemia (AML). Given the known association between -7/del(7q) and RAS pathway mutations, we mined cancer genome databases and explicitly linked CUX1 mutations with oncogenic RAS mutations. To determine if activated RAS and CUX1 deficiency promote leukemogenesis, we generated mice bearing NrasG12D and CUX1-knockdown which developed AML, not seen in mice with either mutation alone. Oncogenic RAS imparts increased self-renewal on CUX1-deficient hematopoietic stem/progenitor cells (HSPCs). Reciprocally, CUX1 knockdown amplifies RAS signaling through reduction of negative regulators of RAS/PI3K signaling. Double mutant HSPCs were responsive to PIK3 or MEK inhibition. Similarly, low expression of CUX1 in primary AML samples correlates with sensitivity to the same inhibitors, suggesting a potential therapy for malignancies with CUX1 inactivation. This work demonstrates an unexpected convergence of an oncogene and tumor suppressor gene on the same pathway.

Regulation of the AGS3·G{alpha}i signaling complex by a seven-transmembrane span receptor.

G-protein signaling modulators (GPSM) play diverse functional roles through their interaction with G-protein subunits. AGS3 (GPSM1) contains four G-protein regulatory motifs (GPR) that directly bind Gα(i) free of Gßγ providing an unusual scaffold for the "G-switch" and signaling complexes, but the mechanism by which signals track into this scaffold are not well understood. We report the regulation of the AGS3·Gα(i) signaling module by a cell surface, seven-transmembrane receptor. AGS3 and Gα(i1) tagged with Renilla luciferase or yellow fluorescent protein expressed in mammalian cells exhibited saturable, specific bioluminescence resonance energy transfer indicating complex formation in the cell. Activation of α(2)-adrenergic receptors or µ-opioid receptors reduced AGS3-RLuc·Gα(i1)-YFP energy transfer by over 30%. The agonist-mediated effects were inhibited by pertussis toxin and co-expression of RGS4, but were not altered by Gßγ sequestration with the carboxyl terminus of GRK2. Gα(i)-dependent and agonist-sensitive bioluminescence resonance energy transfer was also observed between AGS3 and cell-surface receptors typically coupled to Gα(i) and/or Gα(o) indicating that AGS3 is part of a larger signaling complex. Upon receptor activation, AGS3 reversibly dissociates from this complex at the cell cortex. Receptor coupling to both Gαßγ and GPR-Gα(i) offer additional flexibility for systems to respond and adapt to challenges and orchestrate complex behaviors.

Targeting the PIM protein kinases for the treatment of a T-cell acute lymphoblastic leukemia subset.

New approaches are needed for the treatment of patients with T-cell acute lymphoblastic leukemia (T-ALL) who fail to achieve remission with chemotherapy. Analysis of the effects of pan-PIM protein kinase inhibitors on human T-ALL cell lines demonstrated that the sensitive cell lines expressed higher PIM1 protein kinase levels, whereas T-ALL cell lines with NOTCH mutations tended to have lower levels of PIM1 kinase and were insensitive to these inhibitors. NOTCH-mutant cells selected for resistance to gamma secretase inhibitors developed elevated PIM1 kinase levels and increased sensitivity to PIM inhibitors. Gene profiling using a publically available T-ALL dataset demonstrated overexpression of PIM1 in the majority of early T-cell precursor (ETP)-ALLs and a small subset of non-ETP ALL. While the PIM inhibitors blocked growth, they also stimulated ERK and STAT5 phosphorylation, demonstrating that activation of additional signaling pathways occurs with PIM inhibitor treatment. To block these pathways, Ponatinib, a broadly active tyrosine kinase inhibitor (TKI) used to treat chronic myelogenous leukemia, was added to this PIM-inhibitor regimen. The combination of Ponatinib with a PIM inhibitor resulted in synergistic T-ALL growth inhibition and marked apoptotic cell death. Treatment of mice engrafted with human T-ALL with these two agents significantly decreased the tumor burden and improved the survival of treated mice. This dual therapy has the potential to be developed as a novel approach to treat T-ALL with high PIM expression.

Pim1 kinase regulates c-Kit gene translation.

BACKGROUND: Receptor tyrosine kinase, c-Kit (CD117) plays a pivotal role in the maintenance and expansion of hematopoietic stem/progenitor cells (HSPCs). Additionally, over-expression and/or mutational activation of c-Kit have been implicated in numerous malignant diseases including acute myeloid leukemia. However, the translational regulation of c-Kit expression remains largely unknown. METHODS AND RESULTS: We demonstrated that loss of Pim1 led to specific down-regulation of c-Kit expression in HSPCs of Pim1-/- mice and Pim1-/-2-/-3-/- triple knockout (TKO) mice, and resulted in attenuated ERK and STAT3 signaling in response to stimulation with stem cell factor. Transduction of c-Kit restored the defects in colony forming capacity seen in HSPCs from Pim1-/- and TKO mice. Pharmacologic inhibition and genetic modification studies using human megakaryoblastic leukemia cells confirmed the regulation of c-Kit expression by Pim1 kinase: i.e., Pim1-specific shRNA knockdown down-regulated the expression of c-Kit whereas overexpression of Pim1 up-regulated the expression of c-Kit. Mechanistically, inhibition or knockout of Pim1 kinase did not affect the transcription of c-Kit gene. Pim1 kinase enhanced c-Kit 35S methionine labeling and increased the incorporation of c-Kit mRNAs into the polysomes and monosomes, demonstrating that Pim1 kinase regulates c-Kit expression at the translational level. CONCLUSIONS: Our study provides the first evidence that Pim1 regulates c-Kit gene translation and has important implications in hematopoietic stem cell transplantation and cancer treatment.

Activation of Pim Kinases Is Sufficient to Promote Resistance to MET Small-Molecule Inhibitors.

Mesenchymal-epithelial transition (MET) blockade offers a new targeted therapy particularly in those cancers with MET amplification. However, the efficacy and the duration of the response to MET inhibitors are limited by the emergence of drug resistance. Here, we report that resistance to small-molecule inhibitors of MET can arise from increased expression of the prosurvival Pim protein kinases. This resistance mechanism was documented in non-small cell lung cancer and gastric cancer cells with MET amplification. Inhibition of Pim kinases enhanced cell death triggered by short-term treatment with MET inhibitors. Pim kinases control the translation of antiapoptotic protein Bcl-2 at an internal ribosome entry site and this mechanism was identified as the basis for Pim-mediated resistance to MET inhibitors. Protein synthesis was increased in drug-resistant cells, secondary to a Pim-mediated increase in cap-independent translation. In cells rendered drug resistant by chronic treatment with MET inhibitors, genetic or pharmacologic inhibition of Pim kinases was sufficient to restore sensitivity in vitro and in vivo. Taken together, our results rationalize Pim inhibition as a strategy to augment responses and blunt acquired resistance to MET inhibitors in cancer.

Thioredoxin and hematologic malignancies.

Thioredoxin (Trx) is an inflammation-inducible small oxidoreductase protein ubiquitously expressed in all organisms. Trx acts both intracellularly and extracellularly and is involved in a wide range of physiological cellular responses. Inside the cell, Trx alleviates oxidative stress by scavenging reactive oxygen species (ROS), regulates a variety of redox-sensitive signaling pathways as well as ROS-independent genes, and exerts cytoprotective effects. Outside the cell, Trx acts as growth factors or cytokines and promotes cell growth and many other cellular responses. Trx is also implicated in tumorigenesis. Trx is a proto-oncogene and is overexpressed in many cancers and correlates with poor prognosis. Trx stimulates cancer cell survival, promotes tumor angiogenesis, and inhibits both spontaneous apoptosis and drug-induced apoptosis. Inhibitors targeting Trx pathway provide a promising therapeutic strategy for cancer prevention and intervention. More recently, data from our laboratory demonstrate an important role of Trx in expanding long-term repopulating hematopoietic stem cells. In this chapter, we first provide an overview of Trx including its isoforms, compartmentation, and functions. We then discuss the roles of Trx in hematologic malignancies. Finally, we summarize the most recent findings from our lab on the use of Trx to enhance hematopoietic reconstitution following hematopoietic stem cell transplantation.

Using quantitative real-time PCR to determine donor cell engraftment in a competitive murine bone marrow transplantation model.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Abnormal hematopoietic phenotypes in Pim kinase triple knockout mice.

BACKGROUND: Pim (proviral insertion in murine lymphoma) kinases are a small family of constitutively active, highly conservative serine/threonine oncogenic kinases and have 3 members: Pim1, Pim2, and Pim3. Pim kinases are also implicated in the regulation of B- and T- cell responses to cytokines and hematopoietic growth factors. The roles of Pim kinases in the regulation of primitive hematopoietic stem cells (HSCs) are largely unknown. METHODS: In the current study, Pim1-/-2-/-3-/- triple knockout (TKO) mice were used to determine the role of Pim kinases in hematopoiesis. Peripheral blood hematological parameters were measured in Pim TKO mice and age-matched wild-type (WT) controls. Primary, secondary, and competitive transplantations were performed to assay the long-term repopulating HSCs in Pim TKO mice. In vivo BrdU incorporation assay and ex vivo Ki67 staining and caspase 3 labeling were performed to evaluate the proliferation and apoptosis of HSCs in Pim TKO mice. RESULTS: Compared to age-matched WT controls, Pim TKO mice had lower peripheral blood platelet count and exhibited erythrocyte hypochromic microcytosis. The bone marrow cells from Pim TKO mice demonstrated decreased hematopoietic progenitor colony-forming ability. Importantly, Pim TKO bone marrow cells had significantly impaired capacity in rescuing lethally irradiated mice and reconstituting hematopoiesis in primary, secondary and competitive transplant models. In vivo BrdU incorporation in long-term HSCs was reduced in Pim TKO mice. Finally, cultured HSCs from Pim TKO mice showed reduced proliferation evaluated by Ki67 staining and higher rate of apoptosis via caspase 3 activation. CONCLUSIONS: Pim kinases are not only essential in the hematopoietic lineage cell development, but also important in HSC expansion, self-renewal, and long-term repopulation.

Proteomic analysis of murine bone marrow niche microenvironment identifies thioredoxin as a novel agent for radioprotection and for enhancing donor cell reconstitution.

Hematopoiesis is regulated by the bone marrow (BM) niche microenvironment. We recently found that posttransplant administration of AMD3100 (a specific and reversible CXCR4 antagonist) enhanced donor cell engraftment and promoted recovery of all donor cell lineages in a congeneic mouse transplant model. We hypothesized that AMD3100 enhances donor cell reconstitution in part by modulating the levels and constitution of soluble factors in the niche microenvironment. In the current study, the effects of the BM extracellular fluid (supernatant) from AMD3100-treated transplant recipient mice on colony-forming units (CFUs) were examined. A semiquantitative, mass spectrometry-based proteomics approach was used to screen for differentially expressed proteins between the BM supernatants of PBS-treated transplant mice and AMD3100-treated transplant mice. A total of 178 proteins were identified in the BM supernatants. Thioredoxin was among the 32 proteins that displayed greater than a twofold increase in spectral counts in the BM supernatant of AMD3100-treated transplant mice. We found that thioredoxin increased CFUs in a dose-dependent manner. Thioredoxin improved hematopoiesis in irradiated mice and protected mice from radiation-related death. Furthermore, ex vivo exposure to thioredoxin for 24 hours enhanced the long-term repopulation of hematopoietic stem cells. Additionally, combined posttransplant administration of thioredoxin and AMD3100 improved hematologic recovery in primary and secondary transplant recipient mice. Our studies demonstrated that factors in the BM niche microenvironment play a critical role in hematopoiesis. Identifying these factors provides clues on potential novel targets that can be used to enhance hematologic recovery in hematopoietic stem cell transplan`tation.

IKKß regulates the repair of DNA double-strand breaks induced by ionizing radiation in MCF-7 breast cancer cells.

Activation of the IKK-NFκB pathway increases the resistance of cancer cells to ionizing radiation (IR). This effect has been largely attributed to the induction of anti-apoptotic proteins by NFκB. Since efficient repair of DNA double strand breaks (DSBs) is required for the clonogenic survival of irradiated cells, we investigated if activation of the IKK-NFκB pathway also regulates DSB repair to promote cell survival after IR. We found that inhibition of the IKK-NFκB pathway with a specific IKKß inhibitor significantly reduced the repair of IR-induced DSBs in MCF-7 cells. The repair of DSBs was also significantly inhibited by silencing IKKß expression with IKKß shRNA. However, down-regulation of IKKα expression with IKKα shRNA had no significant effect on the repair of IR-induced DSBs. Similar findings were also observed in IKKα and/or IKKß knockout mouse embryonic fibroblasts (MEFs). More importantly, inhibition of IKKß with an inhibitor or down-regulation of IKKß with IKKß shRNA sensitized MCF-7 cells to IR-induced clonogenic cell death. DSB repair function and resistance to IR were completely restored by IKKß reconstitution in IKKß-knockdown MCF-7 cells. These findings demonstrate that IKKß can regulate the repair of DSBs, a previously undescribed and important IKKß kinase function; and inhibition of DSB repair may contribute to cance cell radiosensitization induced by IKKß inhibition. As such, specific inhibition of IKKß may represents a more effective approach to sensitize cancer cells to radiotherapy.

Distribution of activator of G-protein signaling 3 within the aggresomal pathway: role of specific residues in the tetratricopeptide repeat domain and differential regulation by the AGS3 binding partners Gi(alpha) and mammalian inscuteable.

AGS3, a receptor-independent activator of G-protein signaling, is involved in unexpected functional diversity for G-protein signaling systems. AGS3 has seven tetratricopeptide (TPR) motifs upstream of four G-protein regulatory (GPR) motifs that serve as docking sites for Gialpha-GDP. The positioning of AGS3 within the cell and the intramolecular dynamics between different domains of the proteins are likely key determinants of their ability to influence G-protein signaling. We report that AGS3 enters into the aggresome pathway and that distribution of the protein is regulated by the AGS3 binding partners Gialpha and mammalian Inscuteable (mInsc). Gialpha rescues AGS3 from the aggresome, whereas mInsc augments the aggresome-like distribution of AGS3. The distribution of AGS3 to the aggresome is dependent upon the TPR domain, and it is accelerated by disruption of the TPR organizational structure or introduction of a nonsynonymous single-nucleotide polymorphism. These data present AGS3, G-proteins, and mInsc as candidate proteins involved in regulating cellular stress associated with protein-processing pathologies.

The PDZ and band 4.1 containing protein Frmpd1 regulates the subcellular location of activator of G-protein signaling 3 and its interaction with G-proteins.

Activator of G-protein signaling 3 (AGS3) is one of nine mammalian proteins containing one or more G-protein regulatory (GPR) motifs that stabilize the GDP-bound conformation of Galphai. Such proteins have revealed unexpected functional diversity for the "G-switch" in the control of events within the cell independent of the role of heterotrimeric G-proteins as transducers for G-protein-coupled receptors at the cell surface. A key question regarding this class of proteins is what controls their subcellular positioning and interaction with G-proteins. We conducted a series of yeast two-hybrid screens to identify proteins interacting with the tetratricopeptide repeat (TPR) of AGS3, which plays an important role in subcellular positioning of the protein. We report the identification of Frmpd1 (FERM and PDZ domain containing 1) as a regulatory binding partner of AGS3. Frmpd1 binds to the TPR domain of AGS3 and coimmunoprecipitates with AGS3 from cell lysates. Cell fractionation indicated that Frmpd1 stabilizes AGS3 in a membrane fraction. Upon cotransfection of COS7 cells with Frmpd1-GFP and AGS3-mRFP, AGS3-mRFP is observed in regions of the cell cortex and also in membrane extensions or processes where it appears to be colocalized with Frmpd1-GFP based upon the merged fluorescent signals. Frmpd1 knockdown (siRNA) in Cath.a-differentiated neuronal cells decreased the level of endogenous AGS3 in membrane fractions by approximately 50% and enhanced the alpha2-adrenergic receptor-mediated inhibition of forskolin-induced increases in cAMP. The coimmunoprecipitation of Frmpd1 with AGS3 is lost as the amount of Galphai3 in the cell is increased and AGS3 apparently switches its binding partner from Frmpd1 to Galphai3 indicating that the interaction of AGS3 with Frmpd1 and Galphai3 is mutually exclusive. Mechanistically, Frmpd1 may position AGS3 in a membrane environment where it then interacts with Galphai in a regulated manner.