Phosphorylation of DEPDC5, a Component of the GATOR1 Complex, Releases Inhibition of Mtorc1 and Promotes Tumor Growth

Phosphorylation of DEPDC5, a component of the GATOR1 complex, releases inhibition of mTORC1 and promotes tumor growth

Sathish 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 implicated amino acids is regulated by a cascade of protein complexes that as drivers of cancer. Their regulation of tumor growth is closely tied function by modulating the activity of the Rag GTPases, RagA/B to the ability of these enzymes to mainly stimulate protein synthesis and RagC/D (10, 12, 13). The activity of the Rag GTPases is by activating mTORC1 (mammalian target of rapamycin complex 1) repressed by the GATOR1 complex proteins, DEPDC5, NPRL2, signaling, although the exact mechanism is not completely un- and NPRL3, based on GATOR1’s GTPase-activating protein derstood. mTORC1 activity is normally suppressed by amino acid (GAP) activity (14, 15). Since the Rheb GTPase activity is highly starvation through a cascade of multiple regulatory protein com- regulated in tumors by TSC2 phosphorylation (16, 17), we hy- plexes, e.g., GATOR1, GATOR2, and KICSTOR, that reduce the activity pothesized that a similar mechanism could control the GATOR1 of Rag GTPases. Bioinformatic analysis revealed that DEPDC5 (DEP complex and thus Rag GTPase and mTORC1 activity. domain containing protein 5), a component of GATOR1 complex, Here we describe the regulation of GATOR1 complex by the contains Pim and AKT protein kinase phosphorylation consensus phosphorylation of DEPDC5 mediated by the Pim and AKT sequences. DEPDC5 phosphorylation by Pim and AKT kinases was protein kinases. Deleting DEPDC5 or mutating specific phos- CELL BIOLOGY confirmed in cancer cells through the use of phospho-specific anti- phorylation sites within the protein partially blocks the antitumor bodies and transfection of phospho-inactive DEPDC5 mutants. Con- activity of small molecule inhibitors used clinically to inhibit Pim sistent with these findings, during amino acid starvation the and AKT protein kinases (18, 19). These findings shed light on a elevated expression of Pim1 overcame the amino acid inhibitory phosphorylation-dependent regulatory mechanism targeting the protein cascade and activated mTORC1. In contrast, the knockout Pim1/AKT-GATOR1-mTORC1 signaling cascade that is a driver of DEPDC5 partially blocked the ability of small molecule inhibitors of cancer cell proliferation. against Pim and AKT kinases both singly and in combination to suppress tumor growth and mTORC1 activity in vitro and in vivo. Results In animal experiments knocking in a glutamic acid (S1530E) in Pim Kinases Regulate the Amino Acid-Sensitive mTORC1 Pathway. DEPDC5, a phospho mimic, in tumor cells induced a significant level of resistance to Pim and the combination of Pim and AKT inhibitors. Upon amino acid starvation, the GATOR1 protein complex Our results indicate a phosphorylation-dependent regulatory mech- is recruited to the lysosome by the interaction of KICSTOR anism targeting DEPDC5 through which Pim1 and AKT act as up- components after the dissociation of the GATOR2, and this stream effectors of mTORC1 to facilitate proliferation and survival of cancer cells. Significance

Pim kinase | DEPDC5 | GATOR1 | AKT kinase | mTORC1 The mTORC1 (mammalian target of rapamycin complex 1) pathway plays a critical role in driving cancer growth. We have he Pim (proviral integration site for Moloney murine leu- identified a phosphorylation-dependent mechanism that con- Tkemia virus) serine/threonine protein kinases have been im- trols mTORC1 activity in which Pim and AKT kinases, 2 enzymes plicated as a driver of both triple negative breast cancer (TNBC) with increased activity in cancer phosphorylate DEPDC5, a and advanced prostate cancer (1–3). Regulation of tumor growth member of the GATOR1 complex that senses cellular amino by Pim has been closely tied with the ability of this kinase to acid levels. The critical nature of this substrate to the activity of stimulate protein synthesis by activating mTORC1 (mammalian these protein kinases is demonstrated by the fact that deletion target of rapamycin complex 1) signaling (4, 5), but the mechanism or mutation of DEPDC5 partially blocks the ability of Pim and by which Pim regulates mTORC1 signaling is unknown. The in- Pim plus AKT inhibitors to suppress tumor cell growth. Thus, teraction between Pim and AKT kinase pathways has been well protein kinases regulate the amino acid sensing cascade to established and plays an important role in tumorigenesis, as control mTORC1 activity and tumor cell growth. demonstrated by the observation that PI3K/AKT inhibition in- Author contributions: S.K.R.P., A.S.K., and K.O. designed research; S.K.R.P., N.S., and K.O. creases Pim kinase levels thus sustaining mTORC1 activity (6, 7). performed research; S.K.R.P., N.S., J.J.B., V.O., J.H.S., M.C.-V., and K.O. contributed new It has also been shown that tumor resistance to a PI3K/AKT in- reagents/analytic tools; S.K.R.P., N.S., A.S.K., and K.O. analyzed data; and S.K.R.P., A.S.K., hibitor treatment in human breast cancer can be overcome by Pim and K.O. wrote the paper. inhibitor therapy (8), suggesting that Pim and AKT have an The authors declare no conflict of interest. overlapping mechanisms of action. This article is a PNAS Direct Submission. Moreover, mTORC1 controls tumor cell growth, and its activity Published under the PNAS license. is often dysregulated in cancer. This enzyme integrates diverse 1To whom correspondence may be addressed. Email: [email protected] or inputs, including amino acids, growth factors, and stress signals [email protected]. to regulate protein synthesis, autophagy, and nutrient metabolism This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (9–11). mTORC1 activity is controlled by 2 major small GTPases, 1073/pnas.1904774116/-/DCSupplemental. Rheb and Rag. Repression of mTORC1 by the depletion of specific First published September 23, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1904774116 PNAS | October 8, 2019 | vol. 116 | no. 41 | 20505–20510 Downloaded by guest on October 6, 2021 recognizes the motif “RXRXXS*/T*,” demonstrate that Pim1 is A B - Rag WT Rag CA Dox - Dox + capable of phosphorylating DEPDC5 (Fig. 2A). To validate these Leu - 0 2 4 6 02 4 6 h CPAAPCPPA AP C AAP phosphorylation sites, we generated a phospho-specific antibody P-S6K P-S6K T389 T389 against S1002 and purchased a commercially available S1530 an- 1 0.5 1.1 0.5 tibody. The specificity of the antibodies employed for these ex- S6K periments was validated using lysates of 293T cells expressing wild S6K P-S6 type (WT) and site-directed DEPDC5 mutants containing S1002A S240/244 P-S6 and S1530A (Fig. 2 B and C). Both mutant and WT DEPDC5 S6 S240/244 1 0.5 0.3 0 were transfected into 293T cells with and without Pim inhibitor. Pim1 S6 Pim inhibition reduced the phosphorylation of both S1002 and S1530 sites, suggesting that both sites are Pim targets (Fig. 2D). To RagB Tubulin examine AKT activity, we cotransfected the plasmids encoding Tripz Pim1 RagD a constitutively active form of AKT, myristoylated (myr) AKT P-IRS1 S1101 and DEPDC5 into 293T cells. Results demonstrate that AKT P-Foxo3a phosphorylates only the S1530 site on DEPDC5 and not S1002 S318/321 E P-GSK3ß (Fig. 2 ). S9 To examine whether endogenous Pim phosphorylates DEPDC5 GSK3ß in breast tumor cells, BT549 and MDA-MB231 TNBC cell lines Tubulin stably expressing Flag-DEPDC5 were developed. These specific TNBC cell lines were chosen as they have been shown to express Fig. 1. Pim kinases regulate amino acid mediated mTORC1 activation. (A) high levels of the Pim1 kinase (SI Appendix, Fig. S1A) (2, 3). Time course of leucine deprivation (Leu−) with and without Pim1 over- Importantly, the combination of a Pim and an AKT inhibitor expression performed in PC3-LN4 cell line containing Dox-inducible Pim1 (Tripz synergistically decreased the DEPDC5 phosphorylation at the Pim1) vector, treated with and without 100 ng/mL of Dox for 18 h. (B)PC3-LN4 S1530 site, whereas the S1002 site is specifically decreased by Pim control, overexpressing Rag WT or constitutively active Rag (CA) (see SI Ap- inhibitor treatment (Fig. 2F). Both inhibitors were capable of μ pendix, SI Materials and Methods) cells were cultured with DMSO (C), 3 M inhibiting mTORC1, as evidenced by a decrease in P-S6, as well as Pim447 (P), 5 μM GSK690693 (A), and the combination (AP) for 6 h. Cells were β lysed and analyzed by Western blotting (WB). See SI Appendix, SI Materials specific targets IRS1 for Pim and GSK3 for AKT. Since both and Methods for WB quantification. Numerical values shown under the blot AKT and Pim are known to be activated by mitogens (26, 27), we are calculated relative to the DMSO (C) treatment. examined whether a specific growth factor could activate DEPDC5 phosphorylation. The addition of insulin to serum-starved BT-549 cells stably expressing DEPDC5 activates both Pim and AKT ac- complex induces Rag dimers to switch to an inactive conformation tivity, as shown by IRS1 and GSK3β phosphorylation, and stimu- containing GDP-bound RagA/B, thereby inactivating mTORC1 lates a 1.8-fold increase in phosphorylation of DEPDC5 S1530 and (20). To study whether specific protein kinases might also play a a 1.5-fold increase in S1002 (Fig. 2G). To examine whether a role in controlling this pathway, we have chosen to use 3 different specific Pim isoform drives phosphorylation of these sites, Pim1, tumorcelltypes:1)prostatecancer(PC3-LN4);2)breastcancer Pim2, Pim3, and all 3 Pims were knocked down (KD) with siRNA (TNBC; MDA-MB 231 and BT549 and a non-TNBC; HCC1954), in BT549 cells overexpressing DEPDC5. KD of Pim1 alone or all 3 both tumor types have elevated Pim protein kinase and increased Pim isoforms decreased the DEPDC5 phosphorylation (Fig. 2H), AKT activity (2, 3, 7, 21); and 3) the T-ALL (HSB-2), whose while in both BT549 and MDA-MB 231 KD of Pim2 or Pim3 had growth is driven by the Pim kinases without significant AKT input no effect on the DEPDC5 phosphorylation (SI Appendix,Fig.S1B (22). Our results demonstrate that under conditions of leucine and C). To exclude off-target effects of siRNA, we performed starvation of prostate cancer cells, PC3-LN4, Pim1 overexpression rescue experiments using PC3-LN4 cells stably expressing using a doxycycline (Dox)-inducible Pim1 vector sustains mTORC1 DEPDC5 and Dox-inducible Pim1. Pim1 KD was capable of activation as measured by phosphorylation levels of p70S6 kinase inhibiting mTORC1, as evidenced by a decrease in P-S6 and P- on threonine 389 (P-S6K) and ribosomal S6 protein on serine 240/ S6K, as well as decreasing the DEPDC5 phosphorylation and the 244 (P-S6) (Fig. 1A). This indicates that the Pim protein kinase known Pim1 substrate IRS1, although Pim1 KD was not effective could be involved in the regulation of mTORC1 under amino acid- in cells with Dox-induced Pim1 expression (SI Appendix,Fig.S1D). restricted conditions. Using constitutive active forms of Rag B and These results indicate that the mTORC1 suppression caused by D (Rag CA; Rag B 99L and Rag D 77L) GTPases (23) stably Pim1 KD could be rescued by Pim1 overexpression. Thus, Pim1 expressed in PC3-LN4 cells, mTORC1 activity, as measured by P- and AKT regulate the phosphorylation of DEPDC5 as a potential S6K and P-S6, is shown to be resistant to both a Pim kinase in- control mechanism in modulating mTORC1 activity. hibitor, Pim447, and an AKT inhibitor, GSK690693 (Fig. 1B), suggesting the ability of Pim or AKT to regulate mTORC1 is up- DEPDC5 Is Essential for the Pim Kinase-Mediated mTORC1 Regulation. stream of the Rag GTPases. In contrast to P-S6K, the phosphor- Knock out (KO) of DEPDC5 is purported to increase mTORC1 ylation of IRS1 S1101, a substrate of Pim kinases (22), is inhibited activity (25). However, we find that in fresh medium for 6 h the by Pim447 treatment, indicating that Pim kinase activity is sup- DEPDC5 KO effect is minimal compared to parental cells, while pressed by this treatment. Similarly, phosphorylation of GSK3β in nutrient-deprived media either secondary to amino acid de- and Foxo3a, substrates of AKT kinase, is inhibited by GSK690693 pletion or prolonged culture for 72 h, the KO of DEPDC5 has a addition; these data suggest that the Pim and AKT kinases act as much more dramatic effect on mTORC1 activity (SI Appendix, potential upstream regulators of Rag GTPase and play a role in Fig. S2 A and B). Importantly, based on our hypothesis that Pim modulating the signaling mechanism regulated by leucine levels. phosphorylates DEPDC5 and regulates mTORC1 activity, KO of DEPDC5 in PC3-LN4, MDA-MB231, and HSB-2 cell lines Pim and AKT Kinases Phosphorylate DEPDC5 to Regulate mTORC1 develops resistance to genetic or pharmacological inhibition of Signaling. Bioinformatic analysis demonstrates that the GATOR1 Pim kinases, as shown by continued growth and mTORC1 sig- component DEPDC5 contains the Pim consensus phosphorylation naling (Fig. 3 A–D and SI Appendix, Fig. S2A). Additionally, site at S1002 RxRHx[S/T] and contains a potential AKT and Pim when MRKNU1, a breast cancer cell line that does not contain phosphorylation site RxRxx[S/T] at S1530 (15, 24, 25). When DEPDC5 (SI Appendix, Fig. S1A), is transduced with this cDNA, Pim1 is overexpressed in 293T cells, results using an antibody that the cell line then becomes sensitive to Pim dependent inhibition of

20506 | www.pnas.org/cgi/doi/10.1073/pnas.1904774116 Padi et al. Downloaded by guest on October 6, 2021 ABCDDEPDC5 DEPDC5 E -+ + myr-AKT CPim1 DEPDC5 WT SA2 - + ++ + DEPDC5 - - + AZD5363 RXRXXS*/ T* WT SA1 WT - + - + Pim447 -- + - + Pim1 P-DEPDC5 IP --+ Pim447 P-DEPDC5 --- Pim447 + + S1530 Flag IP S1530 P-DEPDC5 P-DEPDC5 P-DEPDC5 Flag DEPDC5 S1002 DEPDC5 S1530 S1002 P

I P-DEPDC5 DEPDC5 Pim1 Pim1 S1002 DEPDC5

CL DEPDC5 WCL Tubulin DEPDC5 AKT P-IRS1 S1101 W

P-IRS1 S1101 L Pim1 P-AKT Actin S473 Tubulin WC Actin Actin F EGFP DEPDC5 G - + + + + + Insulin CAPAP DEPDC5 C PA APCPAAP P-DEPDC5 H P-DEPDC5 S1530 S1530 1 1.8 1.7 1.3 0.4 0.1 C im1/2/3 10.50.50.1 P-DEPDC5 im1 P-DEPDC5 S1002 P P si siN S1002 11.51.51.40.60.6 si P-DEPDC5 10.20.90.2 P-S6 S240/244 S1530 DEPDC5 11.51.500.50 P-DEPDC5 P-S6 S6 S1002 S240/244 DEPDC5 P-GSK3ß S9 S6 P-IRS1 Pim1 P-IRS1 S1101 S1101 IRS1 Pim2 P-GSK3ß S9 DEPDC5 Pim3 GSK3ß AKT Actin Pim1 Actin CELL BIOLOGY Actin BT549 DEPDC5 BT549 BT549 DEPDC5

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 samples were analyzed by WB using the indicated antibodies. (D and E) Plasmids expressing Flag DEPDC5, Pim1, or myr-AKT were cotransfected into 293T cells and then treated with 3 μM Pim447 or the AKT inhibitor AZD5363, 3 μM for 18 h. The Flag-IP samples and WCL were analyzed by WB. (F) BT549 cells stably expressing EGFP as a control or DEPDC5 were incubated with DMSO (C), 3 μMPim447(P),3μM AZD5363 (A), and the combination (AP) for 6 h and cell lysates were analyzed by WB using the indicated antibodies. (G) BT549 cells stably expressing DEPDC5 were starved for 24 h and pretreated with DMSO (C), 3 μM Pim447 (P), 5 μM GSK690693 (A), and the combination (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 cells stably 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, SI Materials and Methods for WB quantification. Numerical values shown are calculated relative to the DMSO (C) treatment for F or no insulin treatment for G.

mTORC1 activity. Because inhibitors in this experiment are added expression of wild-type DEPDC5 in PC3-LN4 and MDA-MB231 in fresh media, major changes in P-S6 levels do not occur with KO cells restores the sensitivity to a Pim inhibitor both alone and DEPDC5 expression (Fig. 3E). Similarly, mTORC1 signaling was in combination with an AKT inhibitor. Thus, these observations not suppressed when PC3-LN4 KO cells are complemented with indicate that the ability of Pim and AKT kinase inhibitors to DEPDC5 (Fig. 3A). These data demonstrate that the ability of Pim control cell growth is partially regulated by DEPDC5. inhibition to block mTORC1 activity is dependent on DEPDC5 levels. DEPDC5 is necessary and sufficient to maintain Pim1- Mutations of the DEPDC5 Phosphorylation Site Alters Sensitivity to dependent mTORC1 activation, and Pim1 is capable of control- Pim and AKT Inhibitor Treatments In Vitro and In Vivo. To test the ling the amino acid-sensing machinery by modifying the GATOR1 ability of DEPDC5 phosphorylation to regulate mTORC1 signal- complex. ing and tumor growth, PC3-LN4 WT, DEPDC5 KO, and CRISPR/Cas9 knockin (KI) of DEPDC5 S1530A (A-MUT, Inhibition of Pim and AKT Kinases Cooperatively Down-Regulates Cell phospho inhibitory), and DEPDC5 S1530E (E-MUT, phospho Growth and the mTORC1 Pathway in a DEPDC5-Dependent Manner. mimic) tumor cells were treated with Pim and AKT inhibitors. mTORC1 activity in both DEPDC5 KO prostate and breast tumor DEPDC5 KO cells were completely resistant to these treatments cells in vitro is resistant to Pim inhibitor (Pimi) and the combi- (SI Appendix,Fig.S4A and B). The S1530E-MUT cells were re- nation of Pim and AKT inhibitor treatment (Fig. 4 A and B and SI sistant to Pim and AKT inhibitor treatment with quite similar cell Appendix,Fig.S3A–C). In comparison, the doses of these inhib- growth and activation of mTORC1 to DEPDC5 KO PC3-LN4 itors were sufficient to block the phosphorylation of other known cells (Fig. 5 A and B). In terms of mTORC1 activity, the PC3- targets of Pim and AKT: IRS1, GSK3β, TSC2, or Foxo3a, re- LN4 DEPDC5 S1002E cells were moderately resistant to both spectively. Additionally, as seen in both cell viability (XTT) and Pim inhibitor treatment and leucine deprivation (SI Appendix, Fig. growth (crystal violet staining or IncuCyte real-time imaging) as- S4C), suggesting possibly that phosphorylation at S1002 might says, the knockout of DEPDC5 also blocked the ability of Pim and affect DEPDC5 along with phosphorylation of S1530 or func- AKT inhibitors to decrease the growth of multiple cancer cells tion independently to regulate other interactions of the DEPDC5 lines, PC3-LN4, BT459, MDA-MB231, and HCC1954 (Fig. 4 C–F protein. Conversely, in PC3-LN4 expressing S1530 A-MUT and SI Appendix,Fig.S3D and E). As shown by cell colony for- mTORC1 signaling is significantly suppressed as detected by P-S6K mation (Fig. 4E) and mTORC1 activity (SI Appendix,Fig.S3F) and P-S6 (Fig. 5C), and growth of these tumor cells is ∼50%

Padi et al. PNAS | October 8, 2019 | vol. 116 | no. 41 | 20507 Downloaded by guest on October 6, 2021 A CTR DEPDC5 KO B CTR DEPDC5 KO C D E CTR DEPDC5 CTR KO 3 DEPDC5 DEPDC5 3 CP CP / CPCP 1 1 CPCPCPCP 1/2/ 120 DEPDC5 P-S6K m1/2 m m m C i i i i P-S6 P P P DEPDC5 P T389 i 100 S240/244 s siNC si si siN si 10.41.10.9 S6 P-S6K DEPDC5 P-S6 80 S240/244 T389 P-S6 Actin S240/244 60 10.21.10.6 MRKNU1 S6K 1 0.6 0.4 S6K 40 P-S6 S6 S240/244 S6 20 P-IRS1 S6 S1101 Viability, % of untreated 0 DEPDC5 P-IRS1 IRS1 HSB-2 CTR DEPDC5 P-IRS1 S1101 KO S1101 Pim1 Tubulin DMSO 1µM IRS1 3µM 5µM PC3-LN4 Pim2 Actin Pim3 HSB-2 Actin MDA-MB231

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 without Flag-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 DEPDC5 KO 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 were incubated 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 was considered 100% and percent growth after individual treatments is reported relative to the DMSO. XTT data shown are the average ± SD of 3 independent experiments. (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 by WB. See SI Appendix, SI Materials and Methods for WB quantification. Numerical values shown are calculated relative to the Si negative control (siNC) treatment for B or 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 by WB. WB data shown are representative of 3 or more independent experiments with similar results.

less both in vitro and in animal experiments when compared to Discussion the wild type (Fig. 5D and SI Appendix,Fig.S4D). However, these Regulation of mTORC1 plays a key role in controlling normal mutant cells were more sensitive to both the AKT inhibitor and and tumorigenic cell growth. The Pim and AKT kinases (24, 31–33) the combination treatment. Thus, the phospho status of S1530 have a dual role in regulating protein synthesis with both kinases plays a role in regulating DEPDC5 and mTORC1 activity. phosphorylating and modifying the activity of various substrates, To clarify why the S1530A cells still responded to Pim and AKT including eIF4B, TSC2, and PRAS40 (5, 17, 34), that control inhibitors, PC3-LN4 WT and DEPDC5 KO cells were treated with critical growth pathways. We demonstrate that by modifying the mTORC1 inhibitor, rapamycin (100 nM). Rapamycin inhibited DEPDC5, these protein kinases stimulate mTORC1 activity and cell growth in WT and KO cells by ∼60%, suggesting that in- control tumor growth. Experiments using overexpression of Pim1 hibition of mTORC1 was not sufficient to totally abrogate cell and AKT protein kinase and a constitutively active Rag mutant growth. However, the addition of Pim and AKT inhibitors along demonstrate that the identical cascade of protein complexes nor- with rapamycin further inhibited cell growth (90%), thus indicating mally regulated by amino acid availability that controls mTORC1 that these kinase inhibitors target other growth pathways in addi- activity is modified by these protein kinases. DEPDC5 contains a tion to mTORC1 (SI Appendix,Fig.S4E), suggesting that these Pim phosphorylation site which was validated by using phospho- inhibitors are functioning similarly in DEPDC5 S1530A KI cells. specific antibodies. The Pim kinase can phosphorylate S1002 and To test the response of growing tumors to dual kinase inhibition, S1530 while AKT phosphorylates S1530. The overlapping ability of PC3-LN4 WT, DEPDC5 KO, and S1530E-MUT KI cells were these 2 protein kinases to phosphorylate DEPDC5 and the im- injected s.c. into male severe combined immunodeficient (SCID) portance of this protein in controlling mTORC1 activity and tumor mice. Once the tumors reached a specific size (200 mm3), mice growth (35–37) may explain the necessity of inhibiting both Pim were treated with Pim447 (30 mg/kg) (3, 28) or AKT inhibitor and AKT activity to block tumor cell growth (6, 24, 32, 38). Our AZD5363 (40 mg/kg) (29, 30), the combination of drugs, or vehicle results demonstrate that DEPDC5 is phosphorylated by Pim1 and once daily by oral gavage without any significant change to the not Pim2 or Pim3. However, KD of Pim1 alone using siRNAs while body weight (SI Appendix,Fig.S5A and B). PC3-LN4 WT xeno- inhibiting DEPDC5 phosphorylation had only a modest effect on grafts responded to the combination kinase inhibitor treatment blocking cell growth, while inhibiting all Pim kinases with siRNAs with significantly decreased tumor growth (P < 0.05), while mice mimicked the effects of the small molecule Pim inhibitor. The ef- with DEPDC5-KO (Fig. 5E) were resistant to these agents, and fect of Pim1 KD alone may be compromised by the presence of DEPDC5 E-MUT xenografts were relatively resistant to the highly active AKT or the phosphorylation by Pim 2, 3 of other combined Pim and AKT inhibitor treatment (Fig. 5 F and G). targets which control cell growth. This observation is a biochemical These results demonstrate that DEPDC5 levels and the phos- example of an isoform-specific substrate for the Pim kinase family phorylation of DEPDC5 on S1530 both play an important role in of enzymes. Previous results from this laboratory demonstrated that the tumor growth inhibitory activity of these anticancer agents. all 3 isoforms can phosphorylate another Pim substrate IRS1 (39). These experiments demonstrate that Pim and AKT protein kinases Our results demonstrate the central importance of the DEPDC5 regulate the activity of GATOR1. Thus, these protein kinases, protein in regulating tumor sensitivity to Pim and AKT inhibitors. which are overexpressed and/or activated in multiple cancers, en- KO of DEPDC5 in breast, prostate, and leukemic cells blocks the hance tumor growth in part by modulating this regulatory mech- ability of Pim and AKT small molecule inhibitors to suppress tu- anism that controls mTORC1 activity. mor growth both in culture and in animal experiments. This result

20508 | www.pnas.org/cgi/doi/10.1073/pnas.1904774116 Padi et al. Downloaded by guest on October 6, 2021 A B C PC3-LN4 D BT549 E PC3-LN4 CTR DEPDC5 KO CTR DEPDC5 KO 120 P<0.05 120 P<0.05 CPAAP C PCAPA AP AP CAPPCAPA AP DEPDC5 DEPDC5 100 100 CTR P-S6 P-S6 80 80 DEPDC5 KO S240/244 S240/244 60 60 DEPDC5 KO S6 S6 DEPDC5 WT P-IRS1 40 40 P-IRS1 S1101 S1101 BT549 20 20 F P-GSK3ß IRS1 CPAAP Viability, % of untreated Viability, % of untreated S9 P-GSK3ß 0 0 P-Foxo3a S9 CTR S318/321 CTR DEPDC5 CTR DEPDC5 GSK3ß KO KO DEPDC5 KO P-TSC2 DMSO Pimi AKTi AKTi+Pimi S939 P-TSC2 S939 TSC2 TSC2 Tubulin Actin

PC3-LN4 MDA-MB231

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 μMPim447(P),5μM GSK690693 (A), and the combination (AP) for 6 h, and cell extracts were subjected to WB. (B) MDA-MB231 CTR and DEPDC5 KO cells were cultured in fresh medium with DMSO (C), 3 μMPim447(P),3μM 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. The percentage 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 μM AZD5363 (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 considered 100% and percent growth after individual treatments is reported relative to the DMSO. XTT data shown are reported as the average ± SD of 3 independent experiments. (E) PC3-LN4 (100 cells/well) CTR, DEPDC5 KO, and KO cells with DEPDC5 overexpression were seeded into 12-well plates and then incubated with either DMSO (C), 3 μMPim447(P),5μM GSK690693 (A), or the combination (AP) for 10 d; every 3 d fresh medium with drugs was added. Colony formation was visualized 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), CELL BIOLOGY 3 μMPim447(P),3μM AZD5363 (A), or the combination (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 are shown. A statistical comparison with Pim inhibitor versus the combination is shown and statistical significance is evaluated using a Student’s t test.

suggests DEPDC5 protein as an important control point in regulating cells. A KI of DEPDC5 changing S1530 to a glutamic acid (E) in the anticancer activity of these inhibitors. Because the S1530 site is PC3-LN4 cells made these cells partially resistant to growth in- modified by both Pim and AKT, its role in controlling the activity of hibition and mTORC1 suppression in vitro and in vivo by the DEPDC5 was further evaluated using PC3-LN4 prostate cancer combination of AKT and Pim inhibitors. Thus, phosphorylation

WT KO S1530E ABC P A AP C PAPACPAPACPAPA Wild type DEPDC5 S1530E Wild type P-S6K F p< 0.05 G KO T389 N.S. 1800 S1530A S6K 1800 p< 0.05 N.S. P-IRS1 1600 1600 S1530E 3 S1101 3 1400 1400 PC3-LN4 DEPDC5 DEPDC5 1200 1200 Tubulin C WT S1530A 1000 1000 C PAPACPAPA Vehicle 800 800 P-S6K D E AKTi+Pimi p<0.05 600 T389 150 150 600 p<0.05

P-S6 Tumor Volume in mm 400 Tumor Volume in mm 400 S240/244 100 200 S6K 100 200 0 0 S6 D25 D54 50 50 D19 D45 DEPDC5 (Treat D0)(Treat D26) (Treat D0) (Treat D29) Tumor Growth % Tubulin Tumor Growth % 0 0 Vehicle Pim447 AZD5363 Pim447+AZD5363 WT SA2 CTR KO

Fig. 5. DEPDC5 phosphorylation by Pim and AKT kinases regulates mTORC1 activity both in vitro and in vivo. (A–C) PC3-LN4 WT, DEPDC5 KO, and PC3-LN4 cells with CRISPR knockin mutants DEPDC5 S1530A and S1530E were seeded into 12-well plates and incubated with DMSO (C), 3 μMPim447(P),5μMGSK-AKTi(A),andthe combination (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 are shown. For WB, cells were cultured in fresh medium with drugs and treated for 6 h, and cell lysates were analyzed by WB using the indicated antibodies. (D)MaleSCID mice were injected s.c. (5 × 106 cells) with PC3-LN4 WT or S1530A MUT (SA2) cells. Mice were monitored for 3 wk and the percentage of tumor growth in S1530A mice (±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 tumors reached 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 of tumor 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-LN4 WT (n = 3) and CRISPR knockin 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 the combination of Pim447 and AZD5363 daily by oral gavage for about 4 wk. The average tumor volume ± SEM was plotted. A statistical comparison with vehicle versus single 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.

Padi et al. PNAS | October 8, 2019 | vol. 116 | no. 41 | 20509 Downloaded by guest on October 6, 2021 of this site plays a role in both mTORC1 regulation and cell The mechanism by which Rag binding to DEPDC5 leads to in- growth. The mutation of S1002E had a less dramatic biologic teraction with an arginine finger on NPRL2 (45) necessary for the effect, suggesting that this site alone may function in concert with GTPase activity is unknown. It is possible that DEPDC5 phos- S1530 to regulate GATOR1. Similar results have been obtained phorylation plays an important role in regulating GTP hydrolysis, previously for eIF4B where S406 was phosphorylated by Pim and and further experiments will be needed to understand how S422 by AKT. However, phosphorylation of both sites is required phosphorylation may control this process. to control translation via the interaction with eIF3 (6, 40). In- Together these experiments define a mechanism by which Pim1 terestingly, we demonstrate that the addition of insulin will in- and AKT kinases function as upstream regulators of mTORC1 crease the phosphorylation of DEPDC5, giving a glimpse into the through DEPDC5 phosphorylation. The results highlight an im- possibility that Rag activity, like Rheb, is controlled by multiple portant rationale for the combination treatment for breast, prostate, factors other than amino acids. Thus, regulation of DEPDC5 and other cancers with Pim and AKT inhibitors and the importance phosphorylation is critical for the antitumor activity of kinase of DEPDC5 levels playing a role in their activity. inhibitors. Rag GTPases interact with mTORC1, GATOR1 components, Materials and Methods and the guanine nucleotide exchange factor (GEF), Ragulator. Rag All in vivo studies were approved by and conducted in accordance with the GTPase interaction with mTORC1 is spatially regulated and the guidelines of the Institutional Animal Care and Use Committees at the Uni- dissociation of Rag can attenuate mTORC1 activity. The inactive versity of Arizona Cancer Center. Detailed materials and methods, including cell form of Rag GTPase can be released from mTORC1 and be culture conditions, cell viability (XTT) assay, cell growth assay, transient trans- reactivated by Ragulator in response to amino acids (41–44). At fection and DNA plasmids, immunoprecipitation, Western blot analysis, mouse least 2 binding modes between Rag GTPases and the GATOR1 xenografts, CRISPR-Cas9 genome editing, information of antibodies and re- complex (25) are needed for mTORC1 to respond to amino acid agents, and statistics are available in SI Appendix, SI Materials and Methods. withdrawal. A strong interaction between the RagA and DEPDC5 blocks the ability of GATOR1 to stimulate GTP hydrolysis, and a ACKNOWLEDGMENTS. We thank Novartis Pharmaceuticals for providing second weaker interaction with NPRL2 and RagA/C stimulates the Pim447. We acknowledge the Experimental Mouse Shared Resource and Genome Editing Core at the University of Arizona Cancer Center (UACC) for helping with GAP activity. In preliminary experiments, phosphorylation of in vivo experiments and CRISPR-Cas9 editing, respectively.Thisresearchwas DEPDC5 did not affect the ability of this protein to interact with supported by UACC support grant P30CA023074, NIH award R01CA173200, and either NPRL2 in the GATOR1 or SZT2 in the KICSTOR complex. Department of Defense award W81XWH-12-1-0560 (to A.S.K.).

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