Phosphorylation of DEPDC5, a component of theGATOR1 complex, releases inhibition of mTORC1and promotes tumor growthSathish K. R. Padia, Neha Singha, Jeremiah J. Bearssb, Virginie Oliveb, Jin H. Songc, Marina Cardó-Vilad,Andrew S. Krafta,b,1, and Koichi Okumurae,1

aUniversity of Arizona Cancer Center, The University of Arizona, Tucson, AZ 85724; bDepartment of Medicine, The University of Arizona, Tucson, AZ 85724;cDepartment of Cellular and Molecular Medicine, The University of Arizona, Tucson, AZ 85724; dDepartment of Otolaryngology, The University of Arizona,Tucson, AZ 85724; and eDepartment of Physiology, The University of Arizona, Tucson, AZ 85724

Edited by Michael N. Hall, University of Basel, Basel, Switzerland, and approved August 28, 2019 (received for review March 20, 2019)

The Pim and AKT serine/threonine protein kinases are implicatedas drivers of cancer. Their regulation of tumor growth is closely tiedto the ability of these enzymes to mainly stimulate protein synthesisby activating mTORC1 (mammalian target of rapamycin complex 1)signaling, although the exact mechanism is not completely un-derstood. mTORC1 activity is normally suppressed by amino acidstarvation through a cascade of multiple regulatory protein com-plexes, e.g., GATOR1, GATOR2, and KICSTOR, that reduce the activityof Rag GTPases. Bioinformatic analysis revealed that DEPDC5 (DEPdomain containing protein 5), a component of GATOR1 complex,contains Pim and AKT protein kinase phosphorylation consensussequences. DEPDC5 phosphorylation by Pim and AKT kinases wasconfirmed in cancer cells through the use of phospho-specific anti-bodies and transfection of phospho-inactive DEPDC5 mutants. Con-sistent with these findings, during amino acid starvation theelevated expression of Pim1 overcame the amino acid inhibitoryprotein cascade and activated mTORC1. In contrast, the knockoutof DEPDC5 partially blocked the ability of small molecule inhibitorsagainst Pim and AKT kinases both singly and in combination tosuppress tumor growth and mTORC1 activity in vitro and in vivo.In animal experiments knocking in a glutamic acid (S1530E) inDEPDC5, a phospho mimic, in tumor cells induced a significant levelof resistance to Pim and the combination of Pim and AKT inhibitors.Our results indicate a phosphorylation-dependent regulatory mech-anism targeting DEPDC5 through which Pim1 and AKT act as up-stream effectors of mTORC1 to facilitate proliferation and survivalof cancer cells.

Pim kinase | DEPDC5 | GATOR1 | AKT kinase | mTORC1

The Pim (proviral integration site for Moloney murine leu-kemia virus) serine/threonine protein kinases have been im-

plicated as a driver of both triple negative breast cancer (TNBC)and advanced prostate cancer (1–3). Regulation of tumor growthby Pim has been closely tied with the ability of this kinase tostimulate protein synthesis by activating mTORC1 (mammaliantarget of rapamycin complex 1) signaling (4, 5), but the mechanismby which Pim regulates mTORC1 signaling is unknown. The in-teraction between Pim and AKT kinase pathways has been wellestablished and plays an important role in tumorigenesis, asdemonstrated by the observation that PI3K/AKT inhibition in-creases Pim kinase levels thus sustaining mTORC1 activity (6, 7).It has also been shown that tumor resistance to a PI3K/AKT in-hibitor treatment in human breast cancer can be overcome by Piminhibitor therapy (8), suggesting that Pim and AKT have anoverlapping mechanisms of action.Moreover, mTORC1 controls tumor cell growth, and its activity

is often dysregulated in cancer. This enzyme integrates diverseinputs, including amino acids, growth factors, and stress signalsto regulate protein synthesis, autophagy, and nutrient metabolism(9–11). mTORC1 activity is controlled by 2 major small GTPases,Rheb and Rag. Repression of mTORC1 by the depletion of specific

amino acids is regulated by a cascade of protein complexes thatfunction by modulating the activity of the Rag GTPases, RagA/Band RagC/D (10, 12, 13). The activity of the Rag GTPases isrepressed by the GATOR1 complex proteins, DEPDC5, NPRL2,and NPRL3, based on GATOR1’s GTPase-activating protein(GAP) activity (14, 15). Since the Rheb GTPase activity is highlyregulated in tumors by TSC2 phosphorylation (16, 17), we hy-pothesized that a similar mechanism could control the GATOR1complex and thus Rag GTPase and mTORC1 activity.Here we describe the regulation of GATOR1 complex by the

phosphorylation of DEPDC5 mediated by the Pim and AKTprotein kinases. Deleting DEPDC5 or mutating specific phos-phorylation sites within the protein partially blocks the antitumoractivity of small molecule inhibitors used clinically to inhibit Pimand AKT protein kinases (18, 19). These findings shed light on aphosphorylation-dependent regulatory mechanism targeting thePim1/AKT-GATOR1-mTORC1 signaling cascade that is a driverof cancer cell proliferation.

ResultsPim Kinases Regulate the Amino Acid-Sensitive mTORC1 Pathway.Upon amino acid starvation, the GATOR1 protein complexis recruited to the lysosome by the interaction of KICSTORcomponents after the dissociation of the GATOR2, and this

Significance

The mTORC1 (mammalian target of rapamycin complex 1)pathway plays a critical role in driving cancer growth. We haveidentified a phosphorylation-dependent mechanism that con-trols mTORC1 activity in which Pim and AKT kinases, 2 enzymeswith increased activity in cancer phosphorylate DEPDC5, amember of the GATOR1 complex that senses cellular aminoacid levels. The critical nature of this substrate to the activity ofthese protein kinases is demonstrated by the fact that deletionor mutation of DEPDC5 partially blocks the ability of Pim andPim plus AKT inhibitors to suppress tumor cell growth. Thus,protein kinases regulate the amino acid sensing cascade tocontrol mTORC1 activity and tumor cell growth.

Author contributions: S.K.R.P., A.S.K., and K.O. designed research; S.K.R.P., N.S., and K.O.performed research; S.K.R.P., N.S., J.J.B., V.O., J.H.S., M.C.-V., and K.O. contributed newreagents/analytic tools; S.K.R.P., N.S., A.S.K., and K.O. analyzed data; and S.K.R.P., A.S.K.,and K.O. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence may be addressed. Email: akraft@uacc.arizona.edu orkoichiok@email.arizona.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1904774116/-/DCSupplemental.

First published September 23, 2019.

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complex induces Rag dimers to switch to an inactive conformationcontaining GDP-bound RagA/B, thereby inactivating mTORC1(20). To study whether specific protein kinases might also play arole in controlling this pathway, we have chosen to use 3 differenttumor cell types: 1) prostate cancer (PC3-LN4); 2) breast cancer(TNBC; MDA-MB 231 and BT549 and a non-TNBC; HCC1954),both tumor types have elevated Pim protein kinase and increasedAKT activity (2, 3, 7, 21); and 3) the T-ALL (HSB-2), whosegrowth is driven by the Pim kinases without significant AKT input(22). Our results demonstrate that under conditions of leucinestarvation of prostate cancer cells, PC3-LN4, Pim1 overexpressionusing a doxycycline (Dox)-inducible Pim1 vector sustains mTORC1activation as measured by phosphorylation levels of p70S6 kinaseon threonine 389 (P-S6K) and ribosomal S6 protein on serine 240/244 (P-S6) (Fig. 1A). This indicates that the Pim protein kinasecould be involved in the regulation of mTORC1 under amino acid-restricted conditions. Using constitutive active forms of Rag B andD (Rag CA; Rag B 99L and Rag D 77L) GTPases (23) stablyexpressed in PC3-LN4 cells, mTORC1 activity, as measured by P-S6K and P-S6, is shown to be resistant to both a Pim kinase in-hibitor, Pim447, and an AKT inhibitor, GSK690693 (Fig. 1B),suggesting the ability of Pim or AKT to regulate mTORC1 is up-stream of the Rag GTPases. In contrast to P-S6K, the phosphor-ylation of IRS1 S1101, a substrate of Pim kinases (22), is inhibitedby Pim447 treatment, indicating that Pim kinase activity is sup-pressed by this treatment. Similarly, phosphorylation of GSK3βand Foxo3a, substrates of AKT kinase, is inhibited by GSK690693addition; these data suggest that the Pim and AKT kinases act aspotential upstream regulators of Rag GTPase and play a role inmodulating the signaling mechanism regulated by leucine levels.

Pim and AKT Kinases Phosphorylate DEPDC5 to Regulate mTORC1Signaling. Bioinformatic analysis demonstrates that the GATOR1component DEPDC5 contains the Pim consensus phosphorylationsite at S1002 RxRHx[S/T] and contains a potential AKT and Pimphosphorylation site RxRxx[S/T] at S1530 (15, 24, 25). WhenPim1 is overexpressed in 293T cells, results using an antibody that

recognizes the motif “RXRXXS*/T*,” demonstrate that Pim1 iscapable of phosphorylating DEPDC5 (Fig. 2A). To validate thesephosphorylation sites, we generated a phospho-specific antibodyagainst S1002 and purchased a commercially available S1530 an-tibody. The specificity of the antibodies employed for these ex-periments was validated using lysates of 293T cells expressing wildtype (WT) and site-directed DEPDC5 mutants containing S1002Aand S1530A (Fig. 2 B and C). Both mutant and WT DEPDC5were transfected into 293T cells with and without Pim inhibitor.Pim inhibition reduced the phosphorylation of both S1002 andS1530 sites, suggesting that both sites are Pim targets (Fig. 2D). Toexamine AKT activity, we cotransfected the plasmids encodinga constitutively active form of AKT, myristoylated (myr) AKTand DEPDC5 into 293T cells. Results demonstrate that AKTphosphorylates only the S1530 site on DEPDC5 and not S1002(Fig. 2E).To examine whether endogenous Pim phosphorylates DEPDC5

in breast tumor cells, BT549 and MDA-MB231 TNBC cell linesstably expressing Flag-DEPDC5 were developed. These specificTNBC cell lines were chosen as they have been shown to expresshigh levels of the Pim1 kinase (SI Appendix, Fig. S1A) (2, 3).Importantly, the combination of a Pim and an AKT inhibitorsynergistically decreased the DEPDC5 phosphorylation at theS1530 site, whereas the S1002 site is specifically decreased by Piminhibitor treatment (Fig. 2F). Both inhibitors were capable ofinhibiting mTORC1, as evidenced by a decrease in P-S6, as well asspecific targets IRS1 for Pim and GSK3β for AKT. Since bothAKT and Pim are known to be activated by mitogens (26, 27), weexamined whether a specific growth factor could activate DEPDC5phosphorylation. The addition of insulin to serum-starved BT-549cells stably expressing DEPDC5 activates both Pim and AKT ac-tivity, as shown by IRS1 and GSK3β phosphorylation, and stimu-lates a 1.8-fold increase in phosphorylation of DEPDC5 S1530 anda 1.5-fold increase in S1002 (Fig. 2G). To examine whether aspecific Pim isoform drives phosphorylation of these sites, Pim1,Pim2, Pim3, and all 3 Pims were knocked down (KD) with siRNAin BT549 cells overexpressing DEPDC5. KD of Pim1 alone or all 3Pim isoforms decreased the DEPDC5 phosphorylation (Fig. 2H),while in both BT549 and MDA-MB 231 KD of Pim2 or Pim3 hadno effect on the DEPDC5 phosphorylation (SI Appendix, Fig. S1 Band C). To exclude off-target effects of siRNA, we performedrescue experiments using PC3-LN4 cells stably expressingDEPDC5 and Dox-inducible Pim1. Pim1 KD was capable ofinhibiting mTORC1, as evidenced by a decrease in P-S6 and P-S6K, as well as decreasing the DEPDC5 phosphorylation and theknown Pim1 substrate IRS1, although Pim1 KD was not effectivein cells with Dox-induced Pim1 expression (SI Appendix, Fig. S1D).These results indicate that the mTORC1 suppression caused byPim1 KD could be rescued by Pim1 overexpression. Thus, Pim1and AKT regulate the phosphorylation of DEPDC5 as a potentialcontrol mechanism in modulating mTORC1 activity.

DEPDC5 Is Essential for the Pim Kinase-Mediated mTORC1 Regulation.Knock out (KO) of DEPDC5 is purported to increase mTORC1activity (25). However, we find that in fresh medium for 6 h theDEPDC5 KO effect is minimal compared to parental cells, whilein nutrient-deprived media either secondary to amino acid de-pletion or prolonged culture for 72 h, the KO of DEPDC5 has amuch more dramatic effect on mTORC1 activity (SI Appendix,Fig. S2 A and B). Importantly, based on our hypothesis that Pimphosphorylates DEPDC5 and regulates mTORC1 activity, KOof DEPDC5 in PC3-LN4, MDA-MB231, and HSB-2 cell linesdevelops resistance to genetic or pharmacological inhibition ofPim kinases, as shown by continued growth and mTORC1 sig-naling (Fig. 3 A–D and SI Appendix, Fig. S2A). Additionally,when MRKNU1, a breast cancer cell line that does not containDEPDC5 (SI Appendix, Fig. S1A), is transduced with this cDNA,the cell line then becomes sensitive to Pim dependent inhibition of

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Fig. 1. Pim kinases regulate amino acid mediated mTORC1 activation. (A)Time course of leucine deprivation (Leu−) with and without Pim1 over-expression performed in PC3-LN4 cell line containing Dox-inducible Pim1 (TripzPim1) vector, treated with and without 100 ng/mL of Dox for 18 h. (B) PC3-LN4control, overexpressing Rag WT or constitutively active Rag (CA) (see SI Ap-pendix, SI Materials and Methods) cells were cultured with DMSO (C), 3 μMPim447 (P), 5 μM GSK690693 (A), and the combination (AP) for 6 h. Cells werelysed and analyzed by Western blotting (WB). See SI Appendix, SI Materialsand Methods for WB quantification. Numerical values shown under the blotare calculated relative to the DMSO (C) treatment.

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mTORC1 activity. Because inhibitors in this experiment are addedin fresh media, major changes in P-S6 levels do not occur withDEPDC5 expression (Fig. 3E). Similarly, mTORC1 signaling wasnot suppressed when PC3-LN4 KO cells are complemented withDEPDC5 (Fig. 3A). These data demonstrate that the ability of Piminhibition to block mTORC1 activity is dependent on DEPDC5levels. DEPDC5 is necessary and sufficient to maintain Pim1-dependent mTORC1 activation, and Pim1 is capable of control-ling the amino acid-sensing machinery by modifying the GATOR1complex.

Inhibition of Pim and AKT Kinases Cooperatively Down-Regulates CellGrowth and the mTORC1 Pathway in a DEPDC5-Dependent Manner.mTORC1 activity in both DEPDC5 KO prostate and breast tumorcells in vitro is resistant to Pim inhibitor (Pimi) and the combi-nation of Pim and AKT inhibitor treatment (Fig. 4 A and B and SIAppendix, Fig. S3 A–C). In comparison, the doses of these inhib-itors were sufficient to block the phosphorylation of other knowntargets of Pim and AKT: IRS1, GSK3β, TSC2, or Foxo3a, re-spectively. Additionally, as seen in both cell viability (XTT) andgrowth (crystal violet staining or IncuCyte real-time imaging) as-says, the knockout of DEPDC5 also blocked the ability of Pim andAKT inhibitors to decrease the growth of multiple cancer cellslines, PC3-LN4, BT459, MDA-MB231, and HCC1954 (Fig. 4 C–Fand SI Appendix, Fig. S3 D and E). As shown by cell colony for-mation (Fig. 4E) and mTORC1 activity (SI Appendix, Fig. S3F)

expression of wild-type DEPDC5 in PC3-LN4 and MDA-MB231KO cells restores the sensitivity to a Pim inhibitor both alone andin combination with an AKT inhibitor. Thus, these observationsindicate that the ability of Pim and AKT kinase inhibitors tocontrol cell growth is partially regulated by DEPDC5.

Mutations of the DEPDC5 Phosphorylation Site Alters Sensitivity toPim and AKT Inhibitor Treatments In Vitro and In Vivo. To test theability of DEPDC5 phosphorylation to regulate mTORC1 signal-ing and tumor growth, PC3-LN4 WT, DEPDC5 KO, andCRISPR/Cas9 knockin (KI) of DEPDC5 S1530A (A-MUT,phospho inhibitory), and DEPDC5 S1530E (E-MUT, phosphomimic) tumor cells were treated with Pim and AKT inhibitors.DEPDC5 KO cells were completely resistant to these treatments(SI Appendix, Fig. S4 A and B). The S1530E-MUT cells were re-sistant to Pim and AKT inhibitor treatment with quite similar cellgrowth and activation of mTORC1 to DEPDC5 KO PC3-LN4cells (Fig. 5 A and B). In terms of mTORC1 activity, the PC3-LN4 DEPDC5 S1002E cells were moderately resistant to bothPim inhibitor treatment and leucine deprivation (SI Appendix, Fig.S4C), suggesting possibly that phosphorylation at S1002 mightaffect DEPDC5 along with phosphorylation of S1530 or func-tion independently to regulate other interactions of the DEPDC5protein. Conversely, in PC3-LN4 expressing S1530 A-MUTmTORC1 signaling is significantly suppressed as detected by P-S6Kand P-S6 (Fig. 5C), and growth of these tumor cells is ∼50%

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Fig. 2. Pim and AKT kinases phosphorylate DEPDC5. (A) Pim1 and Flag-tagged DEPDC5 (Flag DEPDC5) plasmids were cotransfected into 293T cells. Flag immuno-precipitated (IP) samples and whole cell lysates (WCL) were analyzed by WB using the indicated antibodies. (B and C) Plasmids expressing DEPDC5 WT, S1002A (SA1),and S1530A (SA2) were transfected into 293T cells which were treated with and without 3 μM Pim447 for 16 h and then cells were lysed. WCL and Flag-IP sampleswere analyzed by WB using the indicated antibodies. (D and E) Plasmids expressing Flag DEPDC5, Pim1, or myr-AKT were cotransfected into 293T cells and thentreated with 3 μMPim447 or the AKT inhibitor AZD5363, 3 μM for 18 h. The Flag-IP samples andWCL were analyzed byWB. (F) BT549 cells stably expressing EGFP as acontrol or DEPDC5 were incubated with DMSO (C), 3 μM Pim447 (P), 3 μMAZD5363 (A), and the combination (AP) for 6 h and cell lysates were analyzed by WB usingthe indicated antibodies. (G) BT549 cells stably expressing DEPDC5were starved for 24 h and pretreated with DMSO (C), 3 μMPim447 (P), 5 μMGSK690693 (A), and thecombination (AP) for 1.5 h before stimulation with insulin (0.5 μg/mL) for 30 min, and cell lysates were analyzed by WB using the indicated antibodies. (H) BT549 cellsstably expressing DEPDC5 were transfected with siRNA targeting Pim1 or all 3 Pim kinases, and after 48 h, cell lysates were analyzed by WB. See SI Appendix, SIMaterials and Methods for WB quantification. Numerical values shown are calculated relative to the DMSO (C) treatment for F or no insulin treatment for G.

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less both in vitro and in animal experiments when compared tothe wild type (Fig. 5D and SI Appendix, Fig. S4D). However, thesemutant cells were more sensitive to both the AKT inhibitor andthe combination treatment. Thus, the phospho status of S1530plays a role in regulating DEPDC5 and mTORC1 activity.To clarify why the S1530A cells still responded to Pim and AKT

inhibitors, PC3-LN4WT and DEPDC5 KO cells were treated withthe mTORC1 inhibitor, rapamycin (100 nM). Rapamycin inhibitedcell growth in WT and KO cells by ∼60%, suggesting that in-hibition of mTORC1 was not sufficient to totally abrogate cellgrowth. However, the addition of Pim and AKT inhibitors alongwith rapamycin further inhibited cell growth (90%), thus indicatingthat these kinase inhibitors target other growth pathways in addi-tion to mTORC1 (SI Appendix, Fig. S4E), suggesting that theseinhibitors are functioning similarly in DEPDC5 S1530A KI cells.To test the response of growing tumors to dual kinase inhibition,

PC3-LN4 WT, DEPDC5 KO, and S1530E-MUT KI cells wereinjected s.c. into male severe combined immunodeficient (SCID)mice. Once the tumors reached a specific size (200 mm3), micewere treated with Pim447 (30 mg/kg) (3, 28) or AKT inhibitorAZD5363 (40 mg/kg) (29, 30), the combination of drugs, or vehicleonce daily by oral gavage without any significant change to thebody weight (SI Appendix, Fig. S5 A and B). PC3-LN4 WT xeno-grafts responded to the combination kinase inhibitor treatmentwith significantly decreased tumor growth (P < 0.05), while micewith DEPDC5-KO (Fig. 5E) were resistant to these agents, andDEPDC5 E-MUT xenografts were relatively resistant to thecombined Pim and AKT inhibitor treatment (Fig. 5 F and G).These results demonstrate that DEPDC5 levels and the phos-phorylation of DEPDC5 on S1530 both play an important role inthe tumor growth inhibitory activity of these anticancer agents.These experiments demonstrate that Pim and AKT protein kinasesregulate the activity of GATOR1. Thus, these protein kinases,which are overexpressed and/or activated in multiple cancers, en-hance tumor growth in part by modulating this regulatory mech-anism that controls mTORC1 activity.

DiscussionRegulation of mTORC1 plays a key role in controlling normaland tumorigenic cell growth. The Pim and AKT kinases (24, 31–33)have a dual role in regulating protein synthesis with both kinasesphosphorylating and modifying the activity of various substrates,including eIF4B, TSC2, and PRAS40 (5, 17, 34), that controlcritical growth pathways. We demonstrate that by modifyingDEPDC5, these protein kinases stimulate mTORC1 activity andcontrol tumor growth. Experiments using overexpression of Pim1and AKT protein kinase and a constitutively active Rag mutantdemonstrate that the identical cascade of protein complexes nor-mally regulated by amino acid availability that controls mTORC1activity is modified by these protein kinases. DEPDC5 contains aPim phosphorylation site which was validated by using phospho-specific antibodies. The Pim kinase can phosphorylate S1002 andS1530 while AKT phosphorylates S1530. The overlapping ability ofthese 2 protein kinases to phosphorylate DEPDC5 and the im-portance of this protein in controlling mTORC1 activity and tumorgrowth (35–37) may explain the necessity of inhibiting both Pimand AKT activity to block tumor cell growth (6, 24, 32, 38). Ourresults demonstrate that DEPDC5 is phosphorylated by Pim1 andnot Pim2 or Pim3. However, KD of Pim1 alone using siRNAs whileinhibiting DEPDC5 phosphorylation had only a modest effect onblocking cell growth, while inhibiting all Pim kinases with siRNAsmimicked the effects of the small molecule Pim inhibitor. The ef-fect of Pim1 KD alone may be compromised by the presence ofhighly active AKT or the phosphorylation by Pim 2, 3 of othertargets which control cell growth. This observation is a biochemicalexample of an isoform-specific substrate for the Pim kinase familyof enzymes. Previous results from this laboratory demonstrated thatall 3 isoforms can phosphorylate another Pim substrate IRS1 (39).Our results demonstrate the central importance of the DEPDC5

protein in regulating tumor sensitivity to Pim and AKT inhibitors.KO of DEPDC5 in breast, prostate, and leukemic cells blocks theability of Pim and AKT small molecule inhibitors to suppress tu-mor growth both in culture and in animal experiments. This result

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Fig. 3. Pim kinase regulation of mTORC1 activity is dependent on DEPDC5. (A) PC3-LN4 CRISPR-control (CTR) and DEPDC5 KO cells transduced with or withoutFlag-DEPDC5 were cultured in fresh medium with DMSO (C) or 3 μM Pim447 (P) for 6 h. The cell lysates were analyzed by WB. (B) MDA-MB231 CTR and DEPDC5KO cells were transfected with siRNA targeting Pim1 or all 3 Pims, and after 48 h, cells were lysed and analyzed by WB. (C) HSB-2 CTR and DEPDC5 KO cells wereincubated with the indicated doses of Pim447 for 72 h. The percentage of viable cells was measured by an XTT assay. The growth of DMSO control cells wasconsidered 100% and percent growth after individual treatments is reported relative to the DMSO. XTT data shown are the average ± SD of 3 independentexperiments. (D) HSB-2 CTR and DEPDC5 KO cells were cultured in fresh medium with DMSO (C) or 3 μM Pim447 (P) for 8 h, and cell lysates were analyzed byWB.See SI Appendix, SI Materials and Methods for WB quantification. Numerical values shown are calculated relative to the Si negative control (siNC) treatment for Bor control DMSO (C) for C. (E) MRKNU1 cells stably expressing DEPDC5 were incubated with DMSO or 3 μM Pim447 for 6 h and cells were lysed and analyzed byWB. WB data shown are representative of 3 or more independent experiments with similar results.

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suggests DEPDC5 protein as an important control point in regulatingthe anticancer activity of these inhibitors. Because the S1530 site ismodified by both Pim and AKT, its role in controlling the activity ofDEPDC5 was further evaluated using PC3-LN4 prostate cancer

cells. A KI of DEPDC5 changing S1530 to a glutamic acid (E) inPC3-LN4 cells made these cells partially resistant to growth in-hibition and mTORC1 suppression in vitro and in vivo by thecombination of AKT and Pim inhibitors. Thus, phosphorylation

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Fig. 4. DEPDC5 deficiency contributes resistance to Pimi (i, inhibitor) and AKTi. (A) PC3-LN4 CTR and DEPDC5 KO cells were cultured in fresh medium with DMSO(C), 3 μM Pim447 (P), 5 μM GSK690693 (A), and the combination (AP) for 6 h, and cell extracts were subjected to WB. (B) MDA-MB231 CTR and DEPDC5 KO cellswere cultured in fresh medium with DMSO (C), 3 μM Pim447 (P), 3 μM AZD5363 (A), and the combination (AP) for 6 h, and cell lysates were analyzed by WB. (C)PC3-LN4 CTR and DEPDC5 KO cells were cultured with DMSO, 3 μM AZD1208 (Pimi), 5 μM GSK690693 (AKTi), and the combination (AKTi+Pimi) for 72 h. Thepercentage of viable cells was measured by an XTT assay. (D) BT549 CTR and DEPDC5 KO cells were cultured with DMSO, 3 μM Pim447 (Pimi), 3 μMAZD5363 (AKTi),and the combination (AKTi+Pimi) for 72 h. The percentage of viable cells was quantified by an XTT assay. The growth of DMSO control cells was considered100% and percent growth after individual treatments is reported relative to the DMSO. XTT data shown are reported as the average ± SD of 3 independentexperiments. (E) PC3-LN4 (100 cells/well) CTR, DEPDC5 KO, and KO cells with DEPDC5 overexpression were seeded into 12-well plates and then incubated witheither DMSO (C), 3 μM Pim447 (P), 5 μM GSK690693 (A), or the combination (AP) for 10 d; every 3 d fresh medium with drugs was added. Colony formation wasvisualized by crystal violet staining. (F) BT549 (4,000 cells/well) CTR and DEPDC5 KO cells were seeded into 12-well plates and then incubated with either DMSO (C),3 μM Pim447 (P), 3 μMAZD5363 (A), or the combination (AP) for 6 d; culture medium with fresh drugs was replaced every 3 d. Cell growth was visualized by crystalviolet staining and representative images are shown. A statistical comparison with Pim inhibitor versus the combination is shown and statistical significance isevaluated using a Student’s t test.

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Fig. 5. DEPDC5 phosphorylation by Pim and AKT kinases regulates mTORC1 activity both in vitro and in vivo. (A–C) PC3-LN4WT, DEPDC5 KO, and PC3-LN4 cells withCRISPR knockin mutants DEPDC5 S1530A and S1530E were seeded into 12-well plates and incubated with DMSO (C), 3 μM Pim447 (P), 5 μM GSK-AKTi (A), and thecombination (AP) for 6 d. Culture medium with fresh drugs was replaced every 3 d. Cell growth was visualized by crystal violet staining and representative images areshown. ForWB, cells were cultured in freshmediumwith drugs and treated for 6 h, and cell lysates were analyzed byWB using the indicated antibodies. (D) Male SCIDmice were injected s.c. (5 × 106 cells) with PC3-LN4WT or S1530AMUT (SA2) cells. Mice were monitored for 3 wk and the percentage of tumor growth in S1530Amice(±SEM, n = 5) as compared to WT mice is plotted. (E) SCID mice were injected s.c. with 1 × 106 PC3-LN4 CRISPR-CTR or DEPDC5 KO cells in groups of 5. After tumorsreached 200 mm3, mice were treated with vehicle or the combination of Pim447 (30 mg/kg) and AZD5363 (40 mg/kg) daily by oral gavage for 2 wk. The percentage oftumor growth in the treated mice (±SEM) as compared to vehicle control is plotted. (F and G) SCID mice were injected s.c. with 1 × 106 PC3-LN4WT (n = 3) and CRISPRknockin DEPDC5 S1530E mutant (n = 5) cells. After tumors reached 200 mm3, mice were treated with vehicle or Pim447 (30 mg/kg), AZD5363 (40 mg/kg), and thecombination of Pim447 and AZD5363 daily by oral gavage for about 4 wk. The average tumor volume ± SEMwas plotted. A statistical comparison with vehicle versussingle drug and single drug versus the combination treated tumors is shown and statistical significance is evaluated using a Student’s t test. N.S., not significant.

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of this site plays a role in both mTORC1 regulation and cellgrowth. The mutation of S1002E had a less dramatic biologiceffect, suggesting that this site alone may function in concert withS1530 to regulate GATOR1. Similar results have been obtainedpreviously for eIF4B where S406 was phosphorylated by Pim andS422 by AKT. However, phosphorylation of both sites is requiredto control translation via the interaction with eIF3 (6, 40). In-terestingly, we demonstrate that the addition of insulin will in-crease the phosphorylation of DEPDC5, giving a glimpse into thepossibility that Rag activity, like Rheb, is controlled by multiplefactors other than amino acids. Thus, regulation of DEPDC5phosphorylation is critical for the antitumor activity of kinaseinhibitors.Rag GTPases interact with mTORC1, GATOR1 components,

and the guanine nucleotide exchange factor (GEF), Ragulator. RagGTPase interaction with mTORC1 is spatially regulated and thedissociation of Rag can attenuate mTORC1 activity. The inactiveform of Rag GTPase can be released from mTORC1 and bereactivated by Ragulator in response to amino acids (41–44). Atleast 2 binding modes between Rag GTPases and the GATOR1complex (25) are needed for mTORC1 to respond to amino acidwithdrawal. A strong interaction between the RagA and DEPDC5blocks the ability of GATOR1 to stimulate GTP hydrolysis, and asecond weaker interaction with NPRL2 and RagA/C stimulates theGAP activity. In preliminary experiments, phosphorylation ofDEPDC5 did not affect the ability of this protein to interact witheither NPRL2 in the GATOR1 or SZT2 in the KICSTOR complex.

The mechanism by which Rag binding to DEPDC5 leads to in-teraction with an arginine finger on NPRL2 (45) necessary for theGTPase activity is unknown. It is possible that DEPDC5 phos-phorylation plays an important role in regulating GTP hydrolysis,and further experiments will be needed to understand howphosphorylation may control this process.Together these experiments define a mechanism by which Pim1

and AKT kinases function as upstream regulators of mTORC1through DEPDC5 phosphorylation. The results highlight an im-portant rationale for the combination treatment for breast, prostate,and other cancers with Pim and AKT inhibitors and the importanceof DEPDC5 levels playing a role in their activity.

Materials and MethodsAll in vivo studies were approved by and conducted in accordance with theguidelines of the Institutional Animal Care and Use Committees at the Uni-versity of Arizona Cancer Center. Detailedmaterials andmethods, including cellculture conditions, cell viability (XTT) assay, cell growth assay, transient trans-fection and DNA plasmids, immunoprecipitation, Western blot analysis, mousexenografts, CRISPR-Cas9 genome editing, information of antibodies and re-agents, and statistics are available in SI Appendix, SI Materials and Methods.

ACKNOWLEDGMENTS. We thank Novartis Pharmaceuticals for providingPim447.We acknowledge the Experimental Mouse Shared Resource and GenomeEditing Core at the University of Arizona Cancer Center (UACC) for helping within vivo experiments and CRISPR-Cas9 editing, respectively. This research wassupported by UACC support grant P30CA023074, NIH award R01CA173200, andDepartment of Defense award W81XWH-12-1-0560 (to A.S.K.).

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