Protein Kinase D3 Sensitizes RAF Inhibitor RAF265 in Melanoma Cells by Preventing Reactivation of MAPK Signaling

Published OnlineFirst April 28, 2011; DOI: 10.1158/0008-5472.CAN-10-3761

Cancer Tumor and Stem Cell Biology Research

Jian Chen, Qiong Shen, Mark Labow, and L. Alex Gaither

Abstract RAS mutations occur in more than 30% of all human cancers but efforts to directly target mutant RAS signaling as a cancer therapy have yet to succeed. As alternative strategies, RAF and MEK inhibitors have been developed to block oncogenic signaling downstream of RAS. As might be expected, studies of these inhibitors have indicated that tumors with RAS or BRAF mutations display resistance RAF or MEK inhibitors. In order to better understand the mechanistic basis for this resistance, we conducted a RNAi-based screen to identify genes that mediated chemoresistance to the RAF kinase inhibitor RAF265 in a BRAF (V600E) mutant melanoma cell line that is resistant to this drug. In this way, we found that knockdown of protein kinase D3 (PRKD3) could enhance cell killing of RAF and MEK inhibitors across multiple melanoma cell lines of various genotypes and sensitivities to RAF265. PRKD3 blockade cooperated with RAF265 to prevent reactivation of the MAPK signaling pathway, interrupt cell cycle progression, trigger apoptosis, and inhibit colony formation growth. Our findings offer initial proof-of-concept that PRKD3 is a valid target to overcome drug resistance being encountered widely in the clinic with RAF or MEK inhibitors. Cancer Res; 71(12); 4280–91. 2011 AACR.

Introduction are found mutationally activated in 30% of all human cancers, with highest prevalence in pancreas (90%), colon (50%), RAS–RAF mitogen-activated protein kinase (MAPK) signal- thyroid (50%), and lung (30%) cancers (1, 2). Although the ing cascade plays a central role in the regulation of cell RAS oncogene has been studied for more than 3 decades, there proliferation and survival, whereas the deregulation of this is no drug on the market that sufficiently inhibits RAS, despite pathway frequently occurs in human cancers (1–3). As muta- extensive efforts to inhibit activated RAS with low molecular tions in RAS or BRAF occur in more than 30% of human weight inhibitors (3, 8). As an alternative therapeutic strategy, cancers, these proteins are very attractive therapeutic targets RAF and MAP/ERK kinase (MEK) inhibitors have been devel- in many cancer types. Among them, BRAF mutations occur in oped to inhibit the pathway downstream of RAS, and only 1 about 7% of human cancers, with highest prevalence in RAF inhibitor Sorafineb has been approved by Food and Drug melanomas (66%) and thyroid (35%–70%) tumors (4, 5). Administration and several inhibitors are still undergoing Interestingly, 80% of all BRAF mutations are concentrated clinical trials (8). However, clinical responses from these on a single substitution of glutamic acid for valine (V600E) reagents are not as effective or durable as expected, drug within the kinase domain (4). Compared with BRAF, muta- resistance frequently occurs in tumors treated with RAF or tions in the 2 RAF isoforms, ARAF and CRAF, are rarely found MEK inhibitors (2, 8). PLX4032 seems to be an effective RAF in human cancers, which is likely due to lower basal kinase inhibitor in malignant melanoma with an overall response activities (6, 7). All 3 RAS isoforms (KRAS, NRAS, and HRAS) rate of 81%, but responsive time from patients ranged from 2 to more than 18 months and this could limit the long-term efficacy of the drug (9). A recent report suggested that Authors' Affiliations: Novartis Institute for BioMedical Research, Inc., upregulation of N-RAS and other RTK signals such as plate- Cambridge, Massachusetts let-derived growth factor receptor b (PDGFRb) are respons- Note: Supplementary data for this article are available at Cancer Research ible for the acquired resistance to PLX4032 (10). Potential Online (http://cancerres.aacrjournals.org/). mechanisms of resistant to RAF or MEK inhibitors in RAS or Corresponding Authors: Jian Chen, Department of Developmental and Molecular Pathways, Novartis Institute for Biomedical Research, Inc., 500 BRAF mutant cancers can be attributed to either coactivation Technology Square, Cambridge, MA 02139. Phone: 617-871-7411; Fax: of parallel or downstream survival pathways prior to drug 617-871-5783; E-mail: [email protected] or L. Alex Gaither, Depart- treatments or compensatory activation of alternative survival ment of Developmental and Molecular Pathways, Novartis Institute for – Biomedical Research, Inc., 500 Technology Square, Cambridge, MA pathways upon drug administration (11 22). In either situa- 02139. Phone: 617-871-7209; Fax: 617-871-5783; E-mail: tion, combinatorial inhibition of multiple survival pathways is [email protected] required to achieve potent antitumor effects. doi: 10.1158/0008-5472.CAN-10-3761 RAS signaling pathway is more complex than a linear 2011 American Association for Cancer Research. RAS–RAF–MEK signaling cascade (1). Activated RAS protein

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Protein Kinase D3 Sensitizes RAF Inhibitor RAF265

can interact with more than 20 effectors including RAF, Materials and Methods phosphatidylinositol 3-kinases (PI3K), RAC, RAL, and phos- pholipase C epsilon to regulate cell proliferation, survival, Kinome siRNA synthetic lethal screen and data analysis and differentiation (2). There are multiple feedback loops The kinome siRNA SMARTpool library directed against 779 known to activate downstreamofRASwhicharedifferen- kinases was purchased from Dharmacon (catalogue no. G- tially regulated depending on the genetic background and 003500). RNAi screens were prepared with 2 compound doses tumor lineage being studied (23, 24). These feedback loops for 12 plates in A2058 cells (6 plates, in duplicate). Briefly, 4 mL can lead to compensatory activation of parallel survival of 206 nmol/L siRNAs in serum-free medium were stamped pathways upon drug treatments, and tumor cells are flexible into each well, 0.03 mL of DharmaFECT 1 in 4 mL serum-free at utilizing the signal pathways for growth and transforma- medium was mixed and added into each well followed by 30- tion, resulting in rapid drug resistance. Clinical reports show minute incubation at room temperature to form a lipid/siRNA that activation of the MAPK pathway was induced via S6K– complex. Then 1,500 cells in 25 mL complete medium were PI3K–RAS signaling in tumor samples from patients treated loaded on top of siRNA–lipid complex. Final concentration of with RAD001, an inhibitor of PI3K pathway (17). In neu- siRNA for each reaction was 25 nmol/L. The cells were roendocrine tumor cells, inhibitor of RAF strongly induced incubated at 37 C with 5% CO2. At 24 hours after siRNA AKT phosphorylation reflecting an activation of PI3K path- transfection, 5 mL of RAF265 was added into each well to make way, and inhibitors of PI3K pathways induced ERK phos- the final concentration of RAF265 at 0.4 mmol/L. Cell viability phorylation indicating MAPK pathway activation (25). Dual was analyzed at 72 hours post-RAF265 treatment by using targeting of both PI3K and MAPK signaling pathways CellTiter-Glo (CTG) Assay (Promega), and data were acquired showed more potent antitumor effect than single treatment by using an Envision (PerkinElmer). From the primary screen, alone (17, 21, 25). the CTG value was normalized by 1-dimensional (1D) normal- Despite the intensive research efforts on RAS signaling, ization scheme based on plate median of screened 384-well our understanding of its regulation is still limited. To plates. The formula for calculating the 1D-normalized value ¼ 0 0 ¼ identify potential modulators of RAS signaling pathways (denoted as x1D)isx1D log x 1D, where x 1D CTG/ and to uncover the molecular mechanisms underlying resis- medianplate CTG. The normalized Z score (NZ) was calculated tance to RAF or MEK inhibitors, we conducted a siRNA for every x1D. The formula used for calculating NZ1D is NZ1D ¼ screen in combination with a RAF inhibitor (RAF265) to [x1D median (x1D)]/MAD(x1D). MAD (x1D) is the median identify genes and/or pathways that sensitize to RAF265 absolute deviation of x1D and is equal to 1.4826 median treatment in a BRAF (V600E) mutant melanoma cell line (|x1D median (x1D)|). (A2058) which is insensitive to RAF265-induced cell death. Additional Materials and Methods are shown in the Sup- By using this approach, we identified protein kinase D3 plementary Data. (PRKD3) that when knocked down could enhance cell killing by RAF265 in A2058. PRKD3 is 1 of 3 members in the protein Results kinase D (PKD) family which includes PRKD1 and PRKD2. The physiologic functions of PRKD3 are not well understood. A kinome synthetic lethal siRNA screen with RAF265 in Similar to PRKD1 and PRKD2, PRKD3 has been shown to A2058 melanoma cells function in protein trafficking and as a histone deacetylase RAF265 is an orally bioavailable small molecule that is kinase (26, 27). One report showed that PRKD3 enhanced known to inhibit CRAF, wild-type BRAF, mutant BRAF CCK-mediated pancreatic amylase secretion via MEK–ERK– (V600E), and VEGF receptor 2 (VEGFR-2) and is currently RSK signaling and this process was activated by GI hormone in phase I clinical trials for advanced melanoma (30, 31). As (28). Another report showed that PRKD3 expression levels other RAF or MEK inhibitors, RAF265 showed less efficacy in were elevated in human prostate cancers compared with tumors with RAS mutations compared with tumors with a normal tissues; PRKD3 overexpression activated AKT and BRAF mutation (31). RAF265 also shows resistance in some ERK in prostate cancer cell linesandpromotedcellgrowth tumors bearing the BRAF V600E mutation such as the and survival (29). Essential role of PRKD3 in prostate cancer melanoma cell line A2058. By using A2058 as the cellular cells is likely due to regulation of MAPK signaling (29). By model, we conducted a synthetic lethal siRNA screen to using A2058 and A375 (RAF265 sensitive cell line) as mel- identify genetic sensitizers of RAF265. The siRNA SMART- anoma cellular models, we showed that PRKD3 inhibition pool kinome library of 779 kinases was used for the screen. cooperates with RAF265 to prevent the reactivation of We chose to run our screen in the presence of 0.4 mmol/L MAPK signaling pathway, induce PARP cleavage and caspase RAF265 where 20% growth inhibition was achieved, leaving a activity, interrupt cell-cycle progression, and inhibit colony suitable window for additional, siRNA-enhanced cellular formation. Finally, we showed that the PRKD3 inhibition toxicity. As positive technical controls, we used siRNAs sensitizes with multiple RAF and MEK inhibitors in a panel targeting Polo-like kinases 1 (PLK1), which is a regulator of melanoma cell lines, suggesting that PRKD3 functionally of mitosis and, when depleted, results in cell lethality (32). interacts with the MAPK signaling pathway. Thus, PRKD3 BRAF siRNAs were used as the biological positive controls provides a potential cancer target to develop effective ther- and luciferase (LUC) siRNAs were used as the negative apeutic strategies to overcome or prevent drug resistance controls. Duplicates were used for each siRNA and NZ were from RAF or MEK inhibitors. calculated. As shown in Figure 1A and B, control LUC siRNA

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centeredatNZof0andPLK1siRNAshaveaNZranging increased more than 2-fold (Fig. 3B). After PRKD3 siRNA from 17 to 28, indicating a robust signal to noise in the treatments, pERK levels were reduced to 0.2 to 0.8, which screens. We considered a primary hit, a gene whose knock- corresponds to a 2- to 20-fold change compared with the down has a minimal effect on cell growth (NZ < 1.5) in the condition without PRKD3 siRNA transfection (Fig. 3B). The absence of RAF265 but enhances cell killing (NZ > 4.5) in the pAKT levels were reduced to 0.4 to 0.6, which corresponds to a presence of a low dose of RAF265 (Fig. 1C). Twelve primary 4- to 6-fold change compared with the condition without hits that met our predefined criteria were identified includ- PRKD3 siRNA transfection (Fig. 3B). In our system, total ERK ing MGC5601, C9ORF96, ALPK1, CDC7, PRKD3, PDGFRB, and AKT levels were not affected by compound treatment or PAPSS2, PIK3R4, RAGE, RPS6KB2, DAPK1, and MAPK11 PRKD3 knockdown (Fig. 3B). Similar results were obtained (Fig. 1C). To further evaluate the sensitizers, we generated with PRKD3 shRNA transfection (Supplementary Fig. S1). dose curves of RAF265 for all the hits with SMARTpool Collectively, we showed that PRKD3 knockdown prevented siRNAs and observed significant IC50 shifts with 4 hits; the reactivation of pAKT and pERK induced by RAF265 ALPK1, MAPK11, PRKD3, and PIK3R4 (Fig. 1D). The mRNA treatments in A2058 cells. knockdown efficiencies of the 4 target genes were confirmed To further validate PRKD3 in modulating MAPK signaling, by quantitative real time PCR (RT-PCR; Fig. 1E). pERK and pAKT levels were analyzed after MEK inhibitor U0126 treatment, with and without PRKD3 knockdown. Sen- PRKD3 inhibition sensitizes RAF265 to kill A2058 sitization of the MEK inhibitor U0126 was observed after melanoma cells PRKD3 knockdown (Supplementary Fig. S2A). The pERK levels To prioritize hits to follow up and to minimize off-target were reduced at 2 hours and were upregulated at 72 hours effects when using a SMARTpool siRNA approach, we tested post-U0126 treatment (Supplementary Fig. S2B and C). After individual siRNAs of the 4 hits for sensitization with RAF265. PRKD3 siRNA transfection in combination with U0126, the On the basis of the mRNA knockdown levels of the target genes, pERK levels were reduced, in particular at high doses of U0126 we selected 2 potent siRNAs from the 4 individual siRNAs for (Supplementary Fig. S2C). Even though pAKT levels were ALPK1, MAPK11, PIK3R4, and PRKD3 (left panels, Fig. 2A–D) slightly reduced with PRKD3 siRNA transfection in combina- and measured sensitization over a RAF265 dose response (right tion with U0126 treatment, pAKT levels relative to total AKT panels, Fig. 2A–D). Among them, PRKD3 was the only gene in levels were not affected (Supplementary Fig. S2C). We have which multiple independent siRNAs resulted in a significant shown that both RAF and MEK inhibitors tested alone were IC50 shift for RAF265 and distinguished itself as the best unable to induce a sustained inhibition of pERK at 72 hours sensitizer identified from this study (Fig. 2D). To confirm after compound treatment. In contrast, we were able to the sensitization observed between PRKD3 siRNAs and restore pERK inhibition in combination with PRKD3 siRNAs. RAF265, we established 3 independent stable cell lines with In addition, MEK and pMEK levels were found to be reduced inducible PRKD3 short-hairpin RNAs (shRNA) expressed in either by PRKD3 siRNA treatment or by RAF265 treatment A2058 cells. The sequences of PRKD3 shRNAs were different alone and further reduced after PRKD3 siRNAs in combination from the PRKD3 siRNAs used in the screen. The 3 PRKD3 with RAF265 (Fig. 3B). shRNAs when induced with DOX resulted in 2- to 8-fold IC50 Because elevated CRAF protein levels contributed to resis- shifts for RAF265 and the sensitization correlated with protein tance to RAF inhibition in a subset of BRAF mutant tumor knockdown efficiencies of PRKD3 shRNAs (Fig. 2E). For the cells, we tested whether CRAF levels were altered by PRKD3 control condition without DOX induction, knockdown of knockdown (15). We observed increased phospho-CRAF PRKD3 protein levels were not observed, and the sensitization (Ser338) after RAF265 treatments (Fig. 3B), which has been was not significant (Fig. 2E). We showed that knockdown of reported for other RAF inhibitors (33). However, PRKD3 siRNA PRKD3 by using multiple siRNAs and shRNAs enhances the cell mediated knockdown had no effect on pCRAF or CRAF levels, killing effects of RAF265 in the resistant melanoma cell line indicating PRKD3 affects pERK levels in a CRAF-independent A2058. manner. We also used genetic approaches to validate the sensitization between PRKD3 inhibition and BRAF but not PRKD3 inhibition sensitizes RAF265 to prevent CRAF. PRKD3 siRNAs in combination with BRAF siRNA reactivation of MAPK signaling, induce PARP treatments resulted in a greater growth inhibition compared cleavage, increase caspase activity, interrupt with any of the single treatment (Fig. 3C). However, neither the cell-cycle progression, and inhibit colony formation combination of PRKD3 and CRAF siRNA treatments nor the in A2058 cells combination of BRAF and CRAF siRNAs showed potent We analyzed pERK and pAKT levels after RAF265 treatment growth inhibition (Fig. 3C). The knockdown efficiencies of with or without PRKD3 knockdown in A2058 cells. Efficient PRKD3, BRAF, and CRAF siRNAs were validated by Western PRKD3 knockdown was achieved by siRNA transfection as blots (Fig. 3C). shown by Western blot (Fig. 3B). RAF265 treatments resulted To further understand the mechanism of PRKD3 knock- in the reduction of pERK levels in a dose-dependent manner at down–induced RAF265 sensitization, we analyzed apoptosis 2 hours post-RAF265 treatment (Fig. 3A). However, at 72 hours markers, PARP cleavage and caspase activity. RAF265 alone post-RAF265 treatment, both pERK and pAKT levels were was not able to induce detectable PARP cleavage or caspase upregulated as shown with control siRNA treatments, pERK activity in A2058 cells (Figs. 3B and 4A). Similar observations levels were increased 1.6- to 4.2-fold and pAKT levels were were reported for MEK inhibitor AZD6244 which was shown

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A B 0 µmol/L RAF265 0.4 µmol/L RAF265

LUC LUC 0 0

BRAF BRAF –5 –5

–10 –10 NZ_rep1

–15 NZ_rep1 –15 PLK1 PLK1 SRMS SRMS –20 –20

–25 PLK1 –25 PLK1 Figure 1. Primary synthetic lethal –25 –20 –15 –10 –5 0 siRNA screens with RAF265 in –25 –20 –15 –10 –5 0 A2058 cells. A, SMARTpool siRNA NZ_rep2 NZ_rep2 screens without RAF265 or B, in the presence of 0.4 mmol/L C RAF265. The Y- and X-axes refer ALPK1 MGC5601 PDGFRB RAGE to the NZ replicate 1 and 2, 3 PIK3R4 C90RF96 PAPSS2 respectively. C, the primary hits 2 MAPK11 selected from the screen. The 1 RPS6KB2 CDC7 Y- and X-axes refer to the 0 DAPK1 averaged NZ in the presence of 0 and 0.4 mmol/L of RAF265, –1 PRKD3 respectively. The primary hits are –2 labeled by their gene symbols. For –3 A–C, LUC, BRAF, and PLK1 siRNA controls are shown in black –4 stars, diamonds, and triangles, –5 LUC respectively. All experimental NZ_mean_0 µmol/L –6 SMARTpool siRNAs are shown in BRAF grey dots. D, dose curves of –7 RAF265 for selected primary hits –8 in A2058. E, relative RNA –9 expression for selected primary hits as measured by RT-PCR. –10 Error bars are shown and indicate SD calculated from 8 independent –10 –9 –8 –7 –6 –5 –4 –3 –2 –1 0 1 experiments. NZ_mean_0.4 µmol/L DE 1.2 1.2

1 1

0.8 0.8

0.6 Luc 0.6 ALPK1_sp 0.4 0.4

Relative viability Relative MAPK11_sp 0.2 PIK3R4_sp 0.2 Relative RNA expression Relative PRKD3_sp 0 0 0.00 0.03 0.30 3.00 Luc

RAF265 (µmol/L) ALPK_sp PIK3R4_spPRKD3_sp MAPK11_sp

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A 1.2 1.2 LUC 1 1.0 ALPK1_01 ALPK1_04 0.8 0.8 0.6 0.6 0.4 0.4

0.2 viability Relative 0.2

Relative RNA expression Relative 0 0.0 3 4 siRNA Luc 0.00 0.31 0.63 1.25 2.50 5.00 10.00 20.00 RAF265 (µmol/L ) ALPK1_01ALPK1_02ALPK1_0ALPK1_0 1.2 B LUC 2 1.0 MAPK11_02 MAPK11_03 1.6 0.8

1.2 0.6

0.8 0.4 Relative viability Relative 0.4 0.2 0 Relative RNA expression Relative 0.0 0.00 0.31 0.63 1.25 2.50 5.00 10.00 20.00 siRNA Luc RAF265 (µmol/L ) C MAPK11_01MAPK11_02MAPK11_03MAPK11_04 1.2 LUC Figure 2. Validation assays for 4 1.4 PIK3R4_02 primary hits identified from siRNA 1.2 1.0 PIK3R4_03 screens in A2058 cells. Relative RNA 1 0.8 expression and dose curves of RAF265 0.8 0.6 after transfection with siRNAs against 0.6 ALPK1 (A), MAPK11 (B), PIK3R4 (C), and 0.4 0.4 PRKD3 (D) are shown. E, RAF265 dose 0.2 viability Relative 0.2 curves for 3 stable cell lines expressing

Relative RNA expression Relative 0 0.0 PRKD3 shRNAs with (left) or without siRNA Luc _03 0.00 0.31 0.63 1.25 2.50 5.00 10.00 20.00 (right) DOX exposure are shown. 3R4 Western blots show PRKD3 protein RAF265 (µmol/L ) PIK3R4_01PIK3R4_02PIK PIK3R4_04 levels with and without DOX induction in 3 stable cell lines. The numbers below D the image blots indicate the 1.2 1.2 LUC quantification of signal intensities 1 1.0 PRKD3_01 normalized against tubulin. Arrow 0.8 PRKD3_02 indicates the PRKD3 protein at the 0.8 predicted molecular weight, an asterix (*) 0.6 0.6 refers to a nonspecific band. Error bars 0.4 0.4 indicate the SDs for 4 replicates at each

0.2 viability Relative 0.2 condition.

Relative RNA expression Relative 0 0.0 0.00 0.31 0.63 1.25 2.50 5.00 10.00 20.00 siRNA Luc RAF265 (µmol/L )

PRKD3_01PRKD3_02PRKD3_03PRKD3_04 E con con –DOX 1.2 +DOX 1.2 PRKD3_1284 PRKD3_1284 1 PRKD3_3045 1 PRKD3_3045 PRKD3_3393 PRKD3_3393 0.8 0.8 0.6 0.6

0.4 0.4

Relative viability Relative 0.2 0.2 0 0 0 0.31 0.63 1.25 2.5 5 10 20 0 0.31 0.63 1.25 2.5 5 10 20

PRKD3 PRKD3 con shRNA con shRNA 1284 3045 3393 1284 3045 3393 PRKD3 PRKD3 * * 0.11 0.23 0.06 1.00

–DOX Tubulin +DOX Tubulin

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A B 2 h post RAF265 72 h post RAF265

con siRNA PRKD3 siRNA con siRNA PRKD3 siRNA 0 0.16 0.63 2.5 10 0 0.16 0.63 2.5 10 0 0.16 0.63 2.5 10 0 0.16 0.63 2.5 10 RAF265 (µmol/L) pERK (202/204) 1.0 1.6 3.5 3.6 4.2 1.2 0.8 0.7 0.4 0.2 ERK pAKT (473) 1.0 2.6 2.6 2.4 2.4 2.2 0.6 0.5 0.4 0.4 AKT

pMEK (Ser217/221) 1.0 0.6 0.7 0.6 0.8 0.5 0.3 0.4 0.5 0.3 MEK 1.0 0.7 0.8 0.8 0.9 0.8 0.5 0.6 0.6 0.4 PARP

cleaved-PARP Cyclin D1 1.0 0.8 0.3 0.2 0.2 1.0 0.4 0.1 0.1 0.1 Tubulin PRKD3 * pcRAF(Ser338) 1.0 0.9 1.2 1.6 2.8 0.9 1.0 1.1 1.2 2.6 cRAF 1.0 0.9 0.80.90.9 0.9 1.1 1.0 1.1 1.0 C X105 5 5 X10 X10 Luc 10 14 Luc 12 Luc PRKD3_01 CRAF_01 CRAF_01 12 10 8 PRKD3_02 CRAF_02 CRAF_02 10 8 6 8 6 6 4 4 Cell viability 4 Cell viability Cell viability 2 2 2 0 0 0 siRNA LuC BRAF_01 BRAF_02 siRNA LuC PRKD3_01 PRKD3_02 siRNA LuC BRAF_01 BRAF_02

PRKD3 BRAF CRAF LUC siRNA LUC LUC 01 02 01 02 01 02 PRKD3 BRAF CRAF * Tubulin Actin Tubulin

Figure 3. PRKD3 inhibition prevents reactivation of pERK and pAKT in A2058 cells. A, Western blots showing protein levels at 2 hours after RAF265 treatment, with and without PRKD3_01 siRNA transfections. B, Western blots showing protein levels at 72 hours after RAF265 treatment, with and without PRKD3_01 siRNA transfections. Tubulin levels served as a loading control. The numbers below the image blots indicate the quantification of signal intensities normalized against tubulin. The signal intensity of 0 mmol/L compound in the absence of PRKD3 knockdown was set as reference 1.0. C, cell viability measured 72 hours after cotransfection with BRAF and PRKD3 siRNAs, CRAF and PRKD3 siRNAs, and BRAF and CRAF siRNAs. Western blots showing protein levels of PRKD3, BRAF, and CRAF after siRNA transfections against PRKD3, BRAF, and CRAF, respectively. Arrow indicates the PRKD3 protein at the predicted molecular weight, an asterix (*) refers to a nonspecific band. Error bars indicate SDs calculated from 4 independent experiments. to induce cell-cycle arrest and growth inhibition rather than activities, indicating PRKD3 knockdown sensitized RAF265 apoptosis in melanoma cell lines and xenograft models (34). to induce apoptosis (Figs. 3B and 4A). PRKD3 siRNA in combination with RAF265 treatments Previous report showed that cyclin D1, a cell-cycle regulator induced significant PARP cleavage and increased caspase for G1 phase entry, was reduced after RAF265 treatments (31).

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A B 70% G0–G1 180,000 Luc 60% 160,000 PRKD3_01 50% 140,000 PRKD3_02 120,000 40% 100,000 30% 80,000 20% Cell numbers 60,000 10% 40,000 Caspase activities 0% 20,000 RAF265 (µmol/L) 00.50 0.5 0 0.5 0 siRNA LUC PRKD3_01 PRKD3_02 RAF265 (µmol/L) 0 0.31 1.25 2.5 5 70% S 60% C 50% RAF265 0 µmol/L 0.5 µmol/L 2 µmol/L 40% 30% 20% Cell numbers 10% Luc 0% RAF265 (µmol/L) 00.500.500.5 siRNA LUC PRKD3_01 PRKD3_02

3.5% G2–M 3.0%

PRKD3_01 2.5% 2.0% 1.5%

Cell numbers 1.0% 0.5% 0.0%

PRKD3_02 RAF265 (µmol/L) 00.50 0.5 0 0.5 siRNA LUC PRKD3_01 PRKD3_02

Figure 4. PRKD3 inhibition sensitizes RAF265 to induce caspase activity, inhibit cell-cycle progression, and colony formation in A2058 cells. A, caspase activity as measured at 72 hours after PRKD3 siRNA transfection. Error bars indicate SDs calculated from 8 independent experiments.

B, cell-cycle profiles of A2058 cells after RAF265 treatment, with and without PRKD3 siRNA transfections. G0–G1,S,andG2–M phases are shown separately. C, colony formation assays in A2058 cells after RAF265 treatment in combination with LUC, PRKD3_01, or PRKD3_02 siRNA transfections.

Cyclin D1 was analyzed in A2058 cells in the presence or phorylated at its activation loops during mitosis, suggesting absence of PRKD3 siRNAs. Consistent with previous reports, that PRKD3 activity is regulated in a cell-cycle dependent RAF265 treatments resulted in a reduction of cyclin D1, and manner (35). Although PRKD3 siRNAs or 0.5 mmol/L RAF265 the reduction was dose dependent (Fig. 3B). PRKD3 siRNA alone resulted in a reduction of G0–G1 cell numbers, we did – alone did not affect cyclin D1 expression, but PRKD3 siRNA not observe cooperative effects in reducing G0 G1 cell num- in combination with RAF265 enhanced the reduction of cyclin bers (Fig. 4B, Supplementary Fig. S3). It is possible that D1, suggesting an interruption of cell-cycle progression the cooperative effects of PRKD3 inhibition and RAF265 in (Fig. 3B). Consistent with this observation, cell-cycle profiles reduction of cyclin D1 is a consequence from blocking of m – showed that PRKD3 siRNAs or 0.5 mol/L RAF265 alone G2 M entry. partially blocked G2–M progression, but PRKD3 siRNAs in Finally, the sensitization of PRKD3 knockdown and combination with 0.5 mmol/L RAF265 completely blocked RAF265 was tested in a colony formation growth assay. – – m G2 M progression, result in a 0% of cells in G2 M (Fig. 4B, PRKD3 siRNAs or RAF265 (0.5 and 2 mol/L) alone partially Supplementary Fig. S3). It was shown that PRKD3 was phos- inhibited the colony formation of A2058 cells, but PRKD3

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siRNAs in combination with 0.5 mmol/L or 2 mmol/L RAF265 PRKD3 inhibition sensitizes with RAF and MEK completely inhibit A2058 cell growth in colonies (Fig. 4C). inhibitors across multiple melanoma cells Taken together, we have shown that PRKD3 knockdown To better understand whether the sensitization between mediates RAF265 sensitization to prevent the reactivation of PRKD3 inhibition and RAF265 in A2058 and A375 cells is MAPK signaling, induce apoptosis markers, reduce cell-cycle shared with other RAF or MEK inhibitors across cell lines of progression, and induce tumor cell growth inhibition at high various lineage and genetic background, we tested additional density in plastic and low density colony formation assays in RAF and MEK inhibitors in a panel of 12 cell lines (Fig. 6 and A2058 cells. Supplementary Fig. S4). In addition to RAF265, we analyzed another RAF inhibitor PLX4032, and 2 MEK inhibitors, U0126 PRKD3 inhibition sensitizes RAF265 to kill the A375 and PD0325901. We tested 12 cell lines including 6 melanoma melanoma cells cell lines carrying BRAF (V600E) mutation (RPMI7951, IGR39, We analyzed a RAF265 sensitive melanoma cell line A375, A2058, A375, SKMEL5, and WM115), 4 cell lines bearing KRAS which also harbors the BRAF (V600E) mutation. A375 cells are mutations of various lineages (PANC1, A549, SW620, and m sensitive to RAF265 with an IC50 of 0.3 mol/L as shown by DU145), 1 nonmelanoma cell line with BRAF (V600E) muta- control LUC siRNAs transfected cells (Fig. 5A). After PRKD3 tion (SKHEP1, liver cancer), and 1 cell line which is wild type siRNA transfection, the IC50 for RAF265 was shifted from 0.3 for both BRAF and RAS (G402, kidney cancer; Fig. 6). mmol/L to 0.16 mmol/L for both PRKD3 siRNAs (Fig. 5A). Next, For both RAF inhibitors RAF265 and PLX4032, 6 of 6 colony formation assays were done. PRKD3 siRNAs or RAF265 melanoma cell lines tested showed sensitization with both (0.05 mmol) alone only partially inhibited A375 cell growth in PRKD3 siRNAs (Fig. 6). Among the lines tested, 3 cell lines colony formation, but PRKD3 siRNAs in combination with 0.05 RPMI7951, IGR39, and A2058 were resistant to RAF265 and mmol/L RAF265 completely inhibited cell growth in colony PLX4032 (Fig. 6A and C) when 2 mmol/L of compound was formation (Fig. 5B). used in combination with PRKD3 siRNAs (Fig. 6B and D). For 3 Similar to A2058 cells, pERK was reactivated after 72 hours sensitive cell lines A375, SKMEL5, and WM115, 0.3 mmol/L of of RAF265 treatment and reduced after the combination with compound was used in the sensitization assays (Fig. 6B and PRKD3 knockdown (Fig. 5D). pMEK and MEK levels were D). Among the cell lines tested, the sensitization between reduced after RAF265 treatment, and further reduced after PRKD3 knockdown and RAF inhibitors was only observed in combination with PRKD3 knockdown (Fig. 5D). In A375, the melanoma cell lines tested, with only 1 exception in which RAF265 resulted in a reduction of cyclin D1, and the reduction PRKD3 siRNAs sensitized with RAF265 in colon cancer cell was dose dependent (Fig. 5C and D). PRKD3 siRNAs alone did line SW620. It could be that additional genetic lesions outside not affect cyclin D1 expression, but PRKD3 siRNA in combi- of the MAPK signaling pathway are necessary for PRKD3 nation with RAF265 enhance the reduction of cyclin D1 sensitization in nonmelanoma lineages. (Fig. 5D). PRKD3 siRNA transfection or RAF265 treatments For both MEK inhibitors, the sensitization was mainly alone in A375 were not sufficient to induce detectable PARP observed with melanoma cell lines (Supplementary Fig. S4). cleavage, whereas PRKD3 siRNA transfection in combination For MEK inhibitor U0126, sensitization with both PRKD3 with RAF265 treatments resulted in significant induction of siRNAs was observed in 5 of 6 melanoma cell lines showed PARP cleavage (Fig. 5C and D). We did not see changes on (Supplementary Fig. S4B), with 1 exception that no sensitiza- total CRAF after RAF265 treatment in A375 cells (Fig. 5D). tion was found with SKMEL5. For MEK inhibitor PD0325901, 4 Instead, we observed decreased phospho-CRAF (Ser338) after of 6 melanoma cell lines exhibited sensitization with both RAF265 treatments (Fig. 5D), it is likely that CRAF is an PRKD3 siRNAs, but sensitization was not found with mela- efficacy target of RAF265 in the A375 cell line. PRKD3 siRNA noma cells IGR39 and WM115 (Supplementary Fig. S4D). In mediated knockdown had no effect on pCRAF or CRAF levels, addition, PRKD3 siRNAs sensitized with PD0325901 in 2 indicating PRKD3 affects pERK levels in a CRAF-independent nonmelanoma cell lines, SW620 and G402. Collectively, we manner. In A375, PRKD3 siRNAs in combination with BRAF showed that PRKD3 inhibition sensitizes with RAF and MEK siRNA treatments resulted in a greater growth inhibition inhibitors in multiple melanoma cell lines. Therefore, it seems compared with any of the single treatments (Fig. 5E). How- that the sensitization between PRKD3 inhibition and RAF or ever, neither the combination of PRKD3 and CRAF siRNA MEK inhibitors is in melanoma lineage selective. treatments nor the combination of BRAF and CRAF siRNAs showed additive growth inhibition (Fig. 5E). The knockdown Discussion efficiencies of PRKD3, BRAF, and CRAF siRNAs were validated by Western blots (Fig. 5E). The effects on levels of pERK, Primary and acquired resistance to RAF and MEK inhibitors pMEK, cyclin D1, and PARP cleavage, as well as colony has been linked to reactivation of the MAPK pathway and formation, after PRKD3 knockdown in combination with PI3K pathway. Rebound of pERK levels has been observed for RAF265 are similar in A375 and A2058 cells, indicating this RAF inhibitor such as PLX4032 and MEK inhibitor like mechanism is shared by different tumor cells from the same PD0325901 (12, 15, 36). Compensatory activation AKT has lineage or with the same genetic background BRAF (V600E). been reported both for the RAF inhibitor RAF265 and MEK These data also suggest that single agent sensitivity to RAF265 inhibitor AZD6244 (AstraZeneca; refs. 20, 25, 37, 38). Here, we is not a requirement for combination PRKD3 knockdown– show that inhibition of PRKD3 sensitizes with RAF265 in a induced cell killing. resistant melanoma cell line (A2058) by preventing rebound of

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AB 0 µmol/L 0.05 µmol/L 0.5 µmol/L RAF265

1,400,000

1,200,000 LUC Luc 1,000,000 PRKD3_01 800,000 PRKD3_02

Viability 600,000 400,000 PRKD3_01 200,000 0 0 0.16 0.31 0.63 1.25 2.5 5 10 Figure 5. PRKD3 inhibition RAF265 (µmol/L) sensitizes RAF265 to kill A375

PRKD3_02 melanoma cells. A, PRKD3 knockdown sensitizes with C 2 h post-RAF265 D 72 h post-RAF265 RAF265 over a dose response con siRNA PRKD3 siRNA con siRNA PRKD3 siRNA treatment as compared with 0 0.16 0.63 2.5 10 0 0.16 0.63 2.5 10 0 0.16 0.63 2.5 0 0.16 0.63 2.5 RAF265 (µmol/L) control LUC siRNAs. Error bars indicate SDs calculated from 8 pERK (202/204) independent experiments. B, 1.0 1.0 2.0 1.8 1.2 1.8 0.7 0.6 colony formation assays in A375 cells after RAF265 treatment, in ERK combination with LUC, 1.0 1.1 1.91.6 1.9 2.5 1.4 1.5 PRKD3_01, or PRKD3_02 siRNA pAKT (473) transfections. Western blots 1.0 1.7 2.4 2.7 0.9 4.6 2.8 2.6 showing protein levels at 2 hours AKT (C) and 72 hours (D) after RAF265 1.11.11.0 0.6 0.7 1.0 0.4 0.2 treatment, with and without PARP PRKD3 siRNA_01 transfection. cleaved-PARP Tubulin levels served as loading controls. The numbers below the Cylin D1 1.0 0.9 0.5 0.2 1.1 0.6 0.1 0.0 image blots indicate the pMEK (217/202) quantification of signal intensities 1.0 0.60.70.8 1.1 0.5 0.4 0.2 normalized against tubulin MEK controls. The signal intensity of m 1.0 0.9 0.8 0.9 1.1 0.9 0.5 0.3 0 mol/L RAF265 in the absence Tubulin of PRKD3 knockdown was then set as reference 1.0. E, cell PRKD3 viability measured at 72 hours * after cotransfection with BRAF pcRAF (338) 1.0 0.4 0.5 0.5 0.9 0.5 0.4 0.4 and PRKD3 siRNAs, CRAF and CRAF PRKD3 siRNAs, and BRAF and 1.0 1.0 1.0 11 0.8 0.8 1.0 1.1 CRAF siRNAs. Western blots E 5 5 X10 5 X10 showing protein levels of PRKD3, X10 Luc 25 Luc Luc BRAF, and CRAF after siRNA 30 CRAF_01 25 PRKD3_01 CRAF_01 transfections against PRKD3, 20 25 CRAF_02 20 PRKD3_02 CRAF_02 20 BRAF, and CRAF, respectively. 15 15 15 10 10 10 Cell viability 5 Cell viability Cell viability 5 5

0 0 0 LuC BRAF_01 BRAF_02 LuC PRKD3_01 PRKD3_02 LuC BRAF_01 BRAF_02

PRKD3 BRAF CRAF LUC siRNA LUC LUC 01 02 01 02 01 02 PRKD3 * BRAF CRAF Tubulin Actin Tubulin

pERK and pAKT. Our data support a compound-induced together with RAF265 in A2058 cells, but not in A375, a resistance mechanism in which the reactivation of the MAPK difference that could be attributable to the presence of the pathway and PI3K pathway act coordinately to promote cell PTEN deletion in the A2058 cells (20). survival when RAF and/or MEK are inhibited. We observed The mechanism of how PRKD3 interacts with pERK and that pAKT was reduced when PRKD3 was knocked down pAKT remains to be clarified. PKCe has been shown to

4288 Cancer Res; 71(12) June 15, 2011 Cancer Research

Protein Kinase D3 Sensitizes RAF Inhibitor RAF265

A RAF265 ) >20

mol/L 15 (µ

50 10 5 0 RPMI7951 IGR39 PANC1 A549 A2058 SW620 A375 SK-MEL5 DU145 G402 WM115 SKHEP1 RAF265 GI B 0 µmol/L RAF265 2 µmol/L RAF265 0 µmol/L RAF265 0.3 µmol/L RAF265 1.5 1.5

1.0 1.0

0.5 0.5 LUC 0.0 0.0

Relative viability Relative RPMI7951 IGR39 PANC1 A549 A2058 SW620 A375 SKMEL5 DU145 G402 WM115 SKHEP1

1.5 1.5 * 1.0 * * * 1.0 ** * 0.5 0.5

0.0 0.0 PRKD3_01 Relative viability Relative RPMI7951 IGR39 PANC1 A549 A2058 SW620 A375 SKMEL5 DU145 G402 WM115 SKHEP1

1.5 1.5

1.0 * * * * 1.0 * * * 0.5 0.5

0.0 0.0 PRKD3_02

Relative viability Relative RPMI7951 IGR39 PANC1 A549 A2058 SW620 A375 SKMEL5 DU145 G402 WM115 SKHEP1 C PLX4032 20

mol/L) 15 µ (

50 10 5

0 IGR39 PANC1 A2058 SW620 G402 A549 SKHEP1 RPMI7951 DU145 SKMEL5 WM115 A375 PLX4032 GI D 0 µmol/L PLX4032 2 µmol/L PLX4032 0 µmol/L PLX4032 0.3 µmol/L PLX4032 1.5 1.5

1.0 1.0 LUC 0.5 0.5 0.0 0.0

Relative viability Relative IGR39 PANC1 A2058 SW620 G402 A549 SKHEP1 RPMI7951 DU145 SKMEL5 WM115 A375

1.5 1.5 1.0 * * * 1.0 * ** 0.5 0.5

0.0 0.0 PRKD3_01

Relative viability Relative IGR39 PANC1 A2058 SW620 G402 A549 SKHEP1 RPMI7951 DU145 SKMEL5 WM115 A375

1.5 1.5 1.0 * * * 1.0 * * * 0.5 0.5 PRKD3_02 0.0 0.0

Relative viability Relative IGR39 PANC1 A2058 SW620 G402 A549 SKHEP1 RPMI7951 DU145 SKMEL5 WM115 A375

Figure 6. PRKD3 inhibition sensitizes RAF inhibitor RAF265 and PLX4032 in multiple melanoma cells. A, GI50s for RAF265 in multiple cell lines (GI50 refers to the concentration of compound that inhibits growth of cells by 50%) not reached up to 20 mmol/L. B, relative cell viabilities after RAF265 treatments, with or without PRKD3 siRNA transfections, are shown for multiple cell lines. C, GI50s for PLX4032 across multiple cell lines. D, relative cell viabilities after PLX4032 treatments, with or without PRKD3 siRNA transfections, are shown for each cell line. Cell viabilities are normalized to 0 mmol/L compound under each condition. Error bars indicate SDs calculated from 6 independent experiments, an asterix (*) refers to a cell line showing sensitization with both PRKD3 siRNAs compared with the control LUC siRNA condition (P < 0.01). www.aacrjournals.org Cancer Res; 71(12) June 15, 2011 4289

Chen et al.

phosphorylate and regulate PRKD3 activity in prostate the ability to clearly define a mechanism of action at this cancer cells (29). We tested whether PKCe knockdown time. can phenocopy the PRKD3 knockdown in sensitizing RAF This study is the first demonstration that PRKD3 inhibition inhibitor. However, we did not observe any sensitization can sensitize with RAF or MEK inhibitors in BRAF (V600E) effects (data not shown). Among the 3 PKD proteins, PRKD1 melanoma cells. Our current data support a model in which is the most extensively studied and has been implicated in a PRKD3 could prevent reactivation of MAPK signaling caused broad range of cellular process and can be activated by a by RAF or MEK inhibitors and sensitize with these inhibitors variety of regulatory peptides (39–41). It has been shown to kill resistant tumor cells. PRKD3 is a potentially druggable that PRKD1 phosphorylates RIN1 and releases it from kinase because there are known inhibitors of this class of competing with RAF for binding to RAS, thus resulting in enzymes (44, 45). It is not clear whether the kinase activity or a an activation of the RAF–MEK–ERK pathway (42). It will be potential scaffold activity of PRKD3 is required for sensitiza- interesting to investigate whether PRKD3 shares any function with the RAF or MEK inhibitor(s). Although our under- tion with PRKD1 such as RIN1 phosphorylation. We have standing of how PRKD3 modulates pERK and pAKT in tested whether PRKD1 or RIN1 siRNAs can sensitize RAF265 response to RAF265 requires further study, PRKD3 provides in killing A2058. We did not observe any sensitization effects a synthetic lethal target opportunity to develop effective (data not shown). In melanoma cells, PRKD2 protein expres- combination therapy to overcome drug-induced resistance sion was undetectable and thus it was not tested in our caused by RAF or MEK inhibitors. sensitization studies. Even though PRKD members share some cellular functions, they can be differentially expressed Disclosure of Potential Conflicts of Interest and exert different functions (28). Further investigation of PRKD3 specific cellular function(s) will be required to better M. Labow and L.A. Gaither are shareholders of Novartis. The other authors understand how PRKD3 ablation augments RAF265 activity disclosed no potential conflicts of interest. in melanoma. One limitation of our screen is that RAF265 can inhibit multiple RAF isoforms, a variety kinases, and Acknowledgments VEGFR (43). Similar to Sorafenib, the efficacy RAF265 in vivo We thank S. Zhao and C. Mickanin for screen reagent preparation, and C. may not be due exclusively to the activity on BRAF (V600E) Voliva, D. Stuart, L. De Parseval, T. Nagel, and P. Finan for advice and discussion. (44). Thus, the sensitizationseenwithPRKD3knockdown The costs of publication of this article were defrayed in part by the payment and RAF265 could be because of a combinatorial effect(s) of page charges. This article must therefore be hereby marked advertisement in with other targets in addition to RAF isoforms and VEGFR. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The lack of selectivity of RAF265 complicates the interpre- Received October 14, 2010; revised April 5, 2011; accepted April 14, 2011; tation of PRKD3 knockdown sensitization and confounds published OnlineFirst April 28, 2011.

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Protein Kinase D3 Sensitizes RAF Inhibitor RAF265 in Melanoma Cells by Preventing Reactivation of MAPK Signaling

Jian Chen, Qiong Shen, Mark Labow, et al.

Cancer Res Published OnlineFirst April 28, 2011.

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