AUM302, a novel triple kinase PIM/PI3K/mTOR inhibitor, is a potent in vitro pancreatic cancer growth inhibitor

Komala Ingle; Joseph F LaComb; Lee M Graves; Antonio T Baines; Agnieszka B Bialkowska

doi:10.1371/journal.pone.0294065

. 2023 Nov 9;18(11):e0294065. doi: 10.1371/journal.pone.0294065

AUM302, a novel triple kinase PIM/PI3K/mTOR inhibitor, is a potent in vitro pancreatic cancer growth inhibitor

Komala Ingle ¹, Joseph F LaComb ¹, Lee M Graves ², Antonio T Baines ^2,³, Agnieszka B Bialkowska ^1,^*

Editor: Wagdy Mohamed Eldehna⁴

PMCID: PMC10635512 PMID: 37943821

Abstract

Pancreatic cancer is one of the leading causes of cancer deaths, with pancreatic ductal adenocarcinoma (PDAC) being the most common subtype. Advanced stage diagnosis of PDAC is common, causing limited treatment opportunities. Gemcitabine is a frequently used chemotherapeutic agent which can be used as a monotherapy or in combination. However, tumors often develop resistance to gemcitabine. Previous studies show that the proto-oncogene PIM kinases (PIM1 and PIM3) are upregulated in PDAC compared to matched normal tissue and are related to chemoresistance and PDAC cell growth. The PIM kinases are also involved in the PI3K/AKT/mTOR pathway to promote cell survival. In this study, we evaluate the effect of the novel multikinase PIM/PI3K/mTOR inhibitor, AUM302, and commercially available PIM inhibitor, TP-3654. Using five human PDAC cell lines, we found AUM302 to be a potent inhibitor of cell proliferation, cell viability, cell cycle progression, and phosphoprotein expression, while TP-3654 was less effective. Significantly, AUM302 had a strong impact on the viability of gemcitabine-resistant PDAC cells. Taken together, these results demonstrate that AUM302 exhibits antitumor activity in human PDAC cells and thus has the potential to be an effective drug for PDAC therapy.

Introduction

Pancreatic cancer is the seventh leading cause of cancer deaths worldwide and the third leading cause of cancer deaths in the United States and its predicated to become the second leading cause of death by 2030 [1, 2]. Pancreatic cancer has a poor prognosis, with a 5-year survival rate of just 12% [1, 3]. This low survival rate is caused by several factors, of which perhaps the most important is the prevalence of late-stage diagnoses, with 80% of patients having locally advanced or metastatic pancreatic cancer at the time of diagnosis [4]. Current treatment options include surgical resection, chemotherapy, and radiotherapy [4]. However, only 10%-20% of patients are eligible for curative resection [5] and tumors often show resistance to chemotherapy and radiotherapy [6, 7]. Gemcitabine is a widely used chemotherapeutic agent against locally advanced and metastatic pancreatic cancer [8–10]. Although pancreatic cancer is most receptive to gemcitabine than other anticancer agents, many patients develop resistance within weeks of starting the treatment [11]. Multiple studies showed that reactivation and/or deregulation of SHH, PI3K/AKT, MEK, WNT, and NOTCH signaling pathways impact cell cycle and apoptosis and, in combination with disruption of gemcitabine metabolism leads to development of gemcitabine resistance [12–18]. Recent studies showed that remodeling of gemcitabine metabolism pathway and targeting apoptotic machinery provide promising results [8, 19–21].

Pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer, is genetically heterogeneous driven by mutations in oncogenes and tumor suppressor. K-Ras mutations are major drivers of PDAC development and progression and are identified in 90% of PDAC cases [22–25]. Mutations in tumor-suppressor genes such as CDKN2A, TP53, or SMAD4, and in oncogenes ERBB2 and EGFR, and in signaling pathways genes and genes regulating metabolism accelerate the formation and progression of pancreatic lesions [23, 24, 26, 27]. Dysregulation of multiple signaling pathways allows tumor cells to resist cell death, increase angiogenesis, invasion and metastasis, modify metabolism to nutrient- and oxygen-deficient environment, and remodel tumor-promoting immune response [25, 28–30].

Proviral integration site for moloney murine leukemia virus kinases (PIMs) are serine/threonine kinases that promote cell survival by regulating the cell cycle, cell proliferation, apoptosis, and transcription [31–33]. The PIM family consists of three members, PIM1, PIM2, and PIM3, from which PIM1 and PIM3 have been shown to be upregulated in solid cancers, while PIM2 mostly in hematological cancers [33–36]. PIM1 is upregulated in primary pancreatic tumor tissue compared to matched normal tissue in PDAC patients due to hypoxic environment and has been identified as prognostic marker [37, 38]. A study by Li and colleagues found PIM3 abundantly expressed in pancreatic cancer tissue but not in normal pancreatic tissue [32]. The overexpression of the PIM kinase family is related to chemotherapy and radiotherapy resistance with PIM3 expression specifically acting as a prognostic indicator related to poor patient survival [39]. PIM1 increases the stability of c-Myc through phosphorylation and together they promote cell cycle progression [40]. Multiple studies demonstrated that selective PIM inhibitors reduce phosphorylation levels of ribosomal protein S6 and thus, modulate the translation potential of numerous cancer cell lines [41–45]. PIM1 and PIM3 can phosphorylate pro-apoptotic BAD at Ser-112 to deactivate it and thereby promote cancer cell survival and progression [40]. Importantly, studies show that inhibition of PIM1 or PMI3 in PDAC cells reduces growth, invasion, and chemosensitizes the cells to gemcitabine treatment, respectively [37, 46]. Furthermore, PIM kinases interact with the PI3K/AKT/mTOR pathway to drive cancer cell proliferation and survival [31, 47]. The regulation of mTOR signaling by PIM can also affect mTOR outputs such as S6 kinase affecting cell growth and metabolism [48]. Consequently, PIM kinases are appropriate targets for cancer therapy through the use of PIM kinase inhibitors.

TP-3654 is a second generation small-molecule PIM kinase inhibitor that has been studied in vitro in several cancers, including pancreatic cancer, and is currently being used in a Phase I first-in-human study in patients with advanced solid tumors [49, 50]. The compound AUM302 is a novel triple PIM/PI3K/mTOR inhibitor that has been shown to induce apoptosis and decrease cell viability in prostate cancer [51]. Co-targeting of PIM and PI3K/AKT/mTOR pathways may be a useful approach as these kinases share several downstream targets such as p21, p27, and BAD [47]. Importantly, PI3K pathway has been implicated as one of the mechanisms of gemcitabine resistance in PDAC and targeting its activity provided potential path to sensitize the cells to gemcitabine [13, 52–56]. The aim of this study is to determine the efficacy of AUM302 in comparison with TP-3654 and gemcitabine in inhibiting pancreatic cancer cell lines growth. Here, we show that AUM302, a novel triple kinase PIM/PI3K/mTOR inhibitor, decreases proliferation of pancreatic cancer cell lines in vitro. Moreover, we showed that AUM302 sensitized pancreatic cancer cells’ response to gemcitabine treatment.

Materials and methods

Cell lines and compounds

PDAC cell lines BxPC-3 (CRL-1687), Capan-2 (HTB-80), MIA PaCa-2 (CRL-1420), PANC-1 (CRL-1469), and Hs766T (HTB-134) were purchased from ATCC (Manassas, VA) in 2020 and 2021. BxPC-3 cells were maintained in RPMI-1640 medium and Capan-2 cells in McCoy’s medium. MIA PaCa-2, Hs766T and PANC-1 cells were maintained in DMEM medium. All media were supplemented with 10% FBS and 1% penicillin/streptomycin. MIA PaCa-2-Gemcitabine (MIA PaCa-2 GemR) resistant cells were a gift from the laboratory of Dr. Lee M. Graves (Department of Pharmacology, School of Medicine, the University of North Carolina at Chapel Hill, Chapel Hill, NC, USA). (MIA PaCa-2 GemR) cells were grown in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin and 50 nM gemcitabine. All cells were maintained at 37°C and 5% CO₂. All experiments were performed on cells with passage range between 5 and 29. TP-3654, GDC-0941, BEZ235, and gemcitabine were purchased from Selleck Chemicals (Houston, TX) and AUM302 was provided by AUM Biosciences. TP-3654, AUM302, GDC-0941, BEZ235 and gemcitabine were suspended in DMSO.

Cell viability assay

BxPC-3, Capan-2, MIA PaCa-2, PANC-1, and Hs766T pancreatic cancer cells were seeded at 1 x 10³ cells per well in 100 μl of appropriate media in 96-well plate format. Twenty-four hours post seeding, cells were treated with DMSO (vehicle) and variable concentrations of TP-3654, AUM302, GDC-0941, BEZ235 and gemcitabine for 72 hours. MIA PaCa-2 GemS and MIA PaCa-2 GemR cell lines were seeded at 1 x 10³ cells per well in 100 μl of appropriate media in 96-well plate format, treated with 10 nM, 100 nM, or 1 μM of TP-3654 or AUM302 for 72 hours. Cell viability was analyzed using the Cell Titer-Glo luciferase assay system (Promega; Madison, WI), according to the manufacturer’s protocol, and a SpectraMax M3 plate reader (Molecular Devices; San Jose, CA). The IC₅₀ values were calculated using GraphPad Prism for Windows version 10.0.2 (GraphPad Software) [57–60].

Cell proliferation assay

BxPC-3, Capan-2, MIA PaCa-2, PANC-1, and Hs766T pancreatic cancer cells were seeded at 7.5 x 10⁴ cells per well in 2 ml of appropriate media in 6-well plate format. Twenty-four hours after seeding, cells were treated with DMSO (vehicle) or TP-3654 (10 nM and 100 nM) or AUM302 (10 nM and 100 nM). MIA PaCa-2 GemR cell line was seeded as mentioned above, and then treated with 10 nM, 100 nM, or 1μM of TP-3654 or AUM302. Cell count was determined using the Z-Series Coulter Counter (Beckman Coulter; Indianapolis, IN) after 24, 48, and 72 hours of treatment. Each experiment was performed in triplicate. The measurement of the control (cells with medium and DMSO) was defined as 100% and the results from other measurements were calculated accordingly [57–60].

Cell cycle assay

BxPC-3, Capan-2, MIA PaCa-2, PANC-1, and Hs766T pancreatic cancer cells were seeded at 7.5 x 10⁴ cells per well in 2 ml of appropriate media in 6-well plate format. Twenty-four hours after seeding, cells were treated with DMSO (vehicle) or TP-3654 (100 nM) or AUM302 (100 nM). Cells were stained with propidium iodide and analyzed by FACS analysis using Cytoflex PC after 24, 48, and 72 hours of treatment. Each experiment was performed in triplicate. The measurement of the control (cells with medium and DMSO) was defined as 100% and the results from other measurements were calculated accordingly [59, 60].

Western blot analysis

BxPC-3, Capan-2, MIA PaCa-2, PANC-1, Hs766T, MIA PaCa-2 GemR pancreatic cancer cells were seeded at 7.5 x 10⁴ cells per well in 2 ml of appropriate media in 6-well plate format. Twenty-four hours post seeding, the first five cell lines were treated with DMSO (vehicle) or TP-3654 (10 and 100 nM) or AUM302 (10 and 100 nM) for 72 hours. MIA PaCa-2 GemR cells were treated with 10 nM, 100 nM, and 1 μM of TP-3654 or AUM302 for 72 hours. Cells were lysed in Laemmli buffer and total protein extracts were subjected to electrophoresis in 4–20% or 10% tris-glycine gels. The proteins were then transferred to a nitrocellulose membrane, blocked in 5% non-fat milk in 1 x TTBS buffer, and developed with appropriate antibodies. Protein bands were detected using an enhanced chemiluminescence detection kit using Azure c400 (Azure Biosystems). Densitometry analysis of western blots was performed using FIJI software [61].

Statistical analysis

The analysis was performed using appropriate statistical test with a value of p < 0.05 considered significant. This analysis was performed using GraphPad Prism for Windows version 10.0.2 (GraphPad Software).

Results

AUM302 is a potent inhibitor of pancreatic cancer cell lines growth in vitro

To assess the efficacy of gemcitabine, TP-3654, and AUM302 on the viability of pancreatic cancer cell lines, we performed cell proliferation and growth assays using BxPC-3, Capan-2, MIA PaCa-2, PANC-1, and Hs766T pancreatic cancer cells treated with variable concentrations of these compounds for 72 hours. Furthermore, we treated these cell lines with two PI3K/mTOR inhibitors, GDC-0941 and BEZ235, to establish whether AUM302 or TP-3654, triple PIM/PI3K/mTOR inhibitors have higher efficacy of growth inhibition than dual-inhibitors [62–64]. We determined IC₅₀ values using the Cell Titer-Glo assay (Fig 1 and Table 1). Our results showed that all five compounds inhibit the viability of tested pancreatic cancer cell lines. However, as shown in Table 1, the AUM302 compound has more favorable IC₅₀ values as compared to gemcitabine, GDC-0941, and BEZ235 in BxPC-3, Capan-2, PANC-1, and Hs766T pancreatic cancer cell lines with AUM302 compound being even more effective than TP-3654. Only in MIA PaCa-2 cell line (Fig 1C, Table 1) are IC₅₀ values for BEZ235 and GDC-0941 lower than AUM302. Further studies compared the effectiveness of two triple PIM/PI3K/mTOR inhibitors: TP-3654 and AUM302.

Fig 1 — Pancreatic cancer cell lines BxPC-3 (A), Capan-2 (B), MIA PaCa-2 (C), PANC-1 (D), and Hs766T (E) were treated with variable concentrations of gemcitabine or BEZ235 or GDC-0941 or TP-3654 or AUM302 twenty-four hours after seeding. Cells were treated with test compounds for 72 hours and cell viability was measured using Cell Titer-Glo. Each experiment was performed in triplicate and the results are shown as mean ±SD (*N = 3*).

Table 1. Experimental analysis of IC₅₀ values of gemcitabine, BEZ235, GDC-0941, TP-3654, and AUM302 tested in BxPC-3, Capan-2, MIA PaCa-2, PANC-1, and Hs766T pancreatic cancer cell lines.

Cell line	Gemcitabine	BEZ235	GDC-0941	TP-3654	AUM302
BxPC-3	1375 ± 1.59	49.96 ± 1.29	418.8 ± 1.18	1418 ± 1.38	41.12 ± 1.07
Capan-2	11080 ± 1.99	1517 ± 12.42	576 ± 1.44	Unstable	376 ± 1.25
MIA PaCa-2	5.40E+09^*	84.84^*	713.4 ± 1.29	40390 ± 3.46	891.2 ± 1.16
PANC-1	341.1 ± 1.38	214.6 ± 1.32	2308 ± 1.60	333.4 ± 1.38	65.6 ± 1.17
Hs766T	1.57E+12^*	1653 ± 3.38	2021 ± 1.52	2494 ± 1.71	182.2 ± 1.16

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The values are expressed in nanomolar concentrations as mean with ± standard error.

*—standard error of mean (SEM) LogIC50 value > 12.

To examine the impact of TP-3654 and AUM302 on the growth of pancreatic cancer cell lines, we performed cell proliferation assay using previously tested pancreatic cancer cell lines. As shown in Fig 2, the two compounds, each tested at 10 nM and 100 nM, significantly inhibited proliferation and growth of pancreatic cancer cells over the course of three-day treatment in comparison to DMSO (vehicle)-treated cells. Additionally, analysis showed that AUM302 demonstrated a robust inhibitory effect, not only in comparison with vehicle but also in comparison to TP-3654 treatment in all tested pancreatic cancer cell lines (Fig 2).

Fig 2 — The following pancreatic cancer cell lines, BxPC-3 (A), Capan-2 (B), MIA PaCa-2 (C), PANC-1 (D), and Hs766T (E), were treated with DMSO or TP-3654 (10 nM and 100 nM), or AUM302 (10 nM and 100 nM). Cell count was determined 24, 48, and 72 hours after treatment using a cell counter. The measurement of the control (cells with DMSO) was defined as 100%. Data represent mean ±SD (*N = 6*). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 calculated with two-way ANOVA.

AUM302 alters cell cycle progression of pancreatic cancer cell lines

PIM kinases and PI3K/mTOR signaling pathways have been shown to play an important role in the regulation of cell cycle progression in multiple cancers, including pancreatic [18, 65–71]. Thus, we evaluated the impact of TP-3654 and AUM302 on cell cycle progression. We treated BxPC-3, Capan-2, MIA PaCa-2, PANC-1, and Hs766T pancreatic cancer cell lines over three days with vehicle or 100 nM of test compounds and then analyzed cell cycle using flow cytometry. Our results demonstrated that TP-3654 at tested concentration (Figs 3–5) does not significantly affect the cell cycle progression of tested pancreatic cancer cell lines. In contrast, AUM302 was able to increase the cell number in G₀/G₁ phases, G₂/M, and decrease the number of cells within the S-phase as compared to vehicle-treated cells. Notably, in the case of AUM302 there was a significant modification in the number of cells within aforementioned phases, in comparison to not only vehicle-treated cells but also to cells treated with TP-3654 compound. Importantly, in BxPC-3 and Capan-2 pancreatic cancer cell lines, treatment with AUM302 compound increased the number of cells within subG₁ population, suggesting that this compound may induce apoptosis (Fig 3). These data suggest that treatment with AUM302 compound alters the cell cycle of pancreatic cancer cell lines and can additionally induce cell apoptosis.

Fig 3 — Cells were treated with DMSO or TP-3654 (100 nM) or AUM302 (100 nM) for 24 (A and D), 48 (B and E), and 72 hours (C and F). Cells were stained with propidium iodide and analyzed by FACS analysis. Data are represented as mean ±SD, *N = 3*, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 calculated with two-way ANOVA.

Fig 5 — Cells were treated with DMSO or TP-3654 (100 nM) or AUM302 (100 nM) for 24 (A), 48 (B), and 72 hours (C). Cells were stained with propidium iodide and analyzed by FACS analysis. Data are represented as mean ±SD, *N = 3*, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 calculated with two-way ANOVA.

Fig 4 — Cells were treated with DMSO or TP-3654 (100 nM) or AUM302 (100 nM) for 24 (A and D), 48 (B and E), and 72 hours (C and F). Cells were stained with propidium iodide and analyzed by FACS analysis. Data are represented as mean ±SD, *N = 3*, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 calculated with two-way ANOVA.

TP-3654 and AUM302 alter the cell signaling pathways controlled by PIM kinases and PI3K/mTOR signaling pathway

PIM kinases have been shown to positively modulate gene expression in the cell cycle and inhibit apoptosis by directly and indirectly regulating multiple targets such as c-Myc, BAD, and P21 [40, 72–75]. Furthermore, studies showed that inhibitors of PIM kinases reduce the phosphorylation status of ribosomal protein S6 in multiple cancer types [76]. In addition, myriad publications showed that the PI3K/mTOR pathway plays an essential role in the tumorigenesis of numerous cancers, including pancreatic cancer [16, 77–79]. Thus, we decided to investigate the ability of TP-3654 and AUM302 to alter the expression levels of the components of these pathways in pancreatic cancer cell lines. We treated BxPC-3, Capan-2, MIA PaCa-2, PANC-1, and Hs766T pancreatic cancer cell lines with DMSO (vehicle) and tested compounds at 10 nM or 100 nM, collecting the cells for western blot analysis at 24h. As shown in Fig 6, TP-3654 and AUM302 alter the expression of analyzed proteins. However, the AUM302 compound has the most negative effect on the phosphorylation of mTOR, AKT, and S6 kinases (Figs 6 and 7) while exerting minimal impact on the level of appropriate total proteins. Additionally, AUM302 significantly inhibited the levels of c-Myc compared to DMSO- and TP-3654 treatments. Thus far, we have demonstrated that AUM302 more effectively inhibits the proliferation of pancreatic cancer cells. Importantly, in contrast to TP-3654, AUM302 significantly modified the progression of the cell cycle and induced apoptosis. It has been shown that PI3K plays a vital role in the development of chemoresistance to gemcitabine in pancreatic cancer, and inhibition of PI3K activity can reverse this resistance [52, 80–83]. Hence, we decided to determine whether treatment with AUM302 inhibits the growth of gemcitabine-resistant pancreatic cancer cells.

Fig 6 — BxPC-3 (A), Capan-2 (B), MIA PaCa-2 (C), PANC-1 (D), and Hs766T (E) cells were treated with DMSO (vehicle) or TP-3654 (10 and 100 nM) or AUM302 (10 and 100 nM) for 24 hours.

Fig 7 — BxPC-3 (A), Capan-2 (B), MIA PaCa-2 (C), PANC-1 (D), and Hs766T (E) cells were treated with DMSO (vehicle) or TP-3654 (10 and 100 nM) or AUM302 (10 and 100 nM) for 24 hours. Each experiment was performed in triplicate and the results are shown as mean ±SD (*N = 3*). Densitometry analysis was performed using FIJI software [61]. Statistical analysis was performed using the Student’s test followed by an analysis of the normal distribution (Tukey’s test). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

AUM302 inhibits growth of gemcitabine-resistant pancreatic cancer cells

To assess the ability of TP-3654 and AUM302 to inhibit the growth of gemcitabine resistant pancreatic cancer cells we employed MIA PaCa-2 Gemcitabine Resistant (MIA PaCa-2 GemR) cell line. MIA PaCa-2 GemR cell line was continually grown in media supplemented with 50 nM gemcitabine. First, we grew MIA PaCa-2 GemS and MIA PaCa-2 GemR cell lines in the presence of TP-3654 and AUM302 at 10 nM, 100 nM, and 1 μM. We assessed the cell viability using Cell Titer-Glo 72 hours post-treatment. As shown in Fig 8, TP-3654 at 10 nM and 100 nM did not affect the growth of MIA PaCa-2 GemS and MIA PaCa-2 GemR cells (Fig 8A and 8B). The significant decrease in MIA PaCa-2 GemR cells viability was shown when cells were treated with 1 μM TP-3654 (Fig 8C). In contrast, even low doses of AUM302 (10 nM and 100 nM) significantly inhibited viability of MIA PaCa-2 GemR (Fig 8D and 8E). The effect was even more pronounced when cells were treated with 1 μM AUM302 (Fig 8F).

Fig 8 — MIA PaCa-2 GemR cell line was treated with 10 nM, 100 nM, or 1 μM of TP-3654 (A, B, & C) or AUM302 (D, E, & F) twenty-four hours after seeding. Cells were treated with test compounds for 72 hours and cell viability was measured using Cell Titer-Glo. Data represents mean ±SD (*N = 6* and *N = 4*). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 calculated with two-way ANOVA.

The effect of TP-3654 and AUM302 was additionally tested on the proliferation of MIA PaCa-2 GemR cell line over three days (Fig 9A). MIA PaCa-2 GemR cells were treated with at DMSO (vehicle) and 10 nM, 100 nM, or 1 μM of TP-3654 and AUM302 and cells were collected and counted 24, 48, and 72 hours post-treatment. As shown in Fig 9A, TP-3654 treatment did not affect the growth of MIA PaCa-2 GemR cells. In contrast, AUM302 reduces the growth of MIA PaCa-2 GemR cells at 72 hours at all tested concentration. Notably, treatment with 1 μM AUM302 almost completely inhibited the growth of MIA PaCa-2 GemR after 24 hours of treatment (Fig 9B). Furthermore, protein analysis was completed on MIA PaCa-2 GemR cells treated with DMSO, or TP-3654 or AUM302 at 10 nM, 100 nM, or 1 μM concentration for 24, 48, and 72 hours before total protein extraction. Western blot analysis demonstrated that components of the PI3K/AKT/mTOR signaling pathway are significantly inhibited with AUM302 compared to DMSO- and TP-3654-treated cells (Fig 9B–9D and S1–S3 Figs). Importantly, the inhibitory effects of AUM302 (100 nM and 1 μM) on the phosphorylation levels of mTOR, AKT, and S6 were noted already at 24-hour timepoint. In addition, the levels of non-phosphorylated counterparts of these proteins were downregulated after the treatment with 1μM AUM302 compared to other treatments. While treatment with TP-3654 resulted with significantly lower inhibition of the phosphorylation of mTOR, AKT, and S6, and downregulation of the total protein levels. Furthermore, we assessed the levels of c-Myc, and we showed that AUM302 at 1 μM concentration was able to significantly reduce its levels at three tested time points.

Discussion

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer often resistant to the widely used chemotherapeutic agent gemcitabine. Currently, limited options exist for the treatment of PDAC. The surgery followed by adjuvant chemotherapy with FOLFIRINOX is available only to 10–15% of PDAC patients and provides an overall survival rate of four and a half years [84–86]. PDAC multidrug treatment provides between two to six months of benefit for patients with locally advanced or metastasis compared to single compound treatment [84, 85]. Consequently, studying PDAC molecular pathways gives rise to targeted therapies that may be a promising approach to treating pancreatic cancer [87]. Targeting PIM kinases has become a novel cancer therapeutic approach [88]. PIM inhibitors such as AZD1208, PIM447, and TP-3654 [16] are used against different cancers and have all entered the clinical stage [89]. A phase I dose-escalation study on 35 patients with solid tumors found that AZD1208 induced no functional response, although the PIM kinases were inhibited [90]. TP-3654 has improved potency, and clinical findings suggest it can treat patients with heavily pretreated, relapsed, and resistant solid tumors. There is a strong rationale to investigate PIM inhibitors in combination with PI3K/AKT/mTOR pathway inhibitors, as their interactions drive cancer cell proliferation and cell survival [31].

PI3K inhibitors are a targeted therapy with limited success in treating PDAC but promising results when combined during pre-clinical studies [16]. Initially, pan-PI3K or PI3K/mTOR inhibitors such as GDC-0941 or BEZ235 were developed and tested in preclinical models. Studies showed that simultaneous inhibition of PI3K with GDC-0941 and MEK or PORCN inhibitors provides a synergistic effect [91–93]. Other studies showed BEZ235 enhanced response to the chemotherapy and antiangiogenic or pan-histone deacetylase inhibitors in PDAC treatment [94, 95]. However, resistance to PI3K inhibitors is possible, and PIM overexpression was found to be related to such resistance [96]. Furthermore, PIM mimics the effects of AKT, leading to cell cycle progression, cell survival, and growth [97]. mTOR is a link between the PIM and PI3K pathways, also responsible for cell survival. These pathways are so intertwined in cancer that a multikinase inhibitor may be an efficient approach.

Here, we investigated using a triple kinase PIM/PI3K/mTOR inhibitor, AUM302, compared to TP-3654 and gemcitabine in PDAC cell lines. Initially, we compared the efficacy of two known dual PI3K/mTOR inhibitors, GDC-0941 and BEZ235, with triple PIM/PI3K/mTOR inhibitors, TP-3654 and AUM302, to reduce the viability of PDAC cell lines. Our results showed AUM302 had lower IC₅₀ values in four tested cell lines (BxPC-3, Capan-2, PANC-1, and Hs766T) than other treatments. MIA PaCa-2 cell line was more susceptible to tested dual inhibitors than triple ones. It has been shown that the downregulation of PIM1 by shRNA in MIA PaCa-2 cell lines decreases proliferation [98]. However, the treatment in the context of PI3K/mTOR inhibitors may not result in more efficient viability inhibition (Fig 1, Table 1). AUM302 potently inhibited growth in PDAC cell lines, generating IC₅₀ values in the low nanomolar range and significantly decreased cell viability. TP-3654 had IC₅₀ values in the micromolar range and was less effective in altering the viability of PDAC cells, and gemcitabine even less so. Follow-up experiments comparing the impact of TP-3654 and AUM302 on cell proliferation and cell cycle showed that AUM302 is a more potent inhibitor of proliferation and blocker of the cell cycle progression than TP-3654 (Figs 2–5). Furthermore, AUM302 consistently decreased the number of cells in the S-phase and increased in G₂/M phase compared to TP-3654 treated cells and control. This may be due to inhibition of PIM1 activity, which regulates normal cell cycle progression, particularly at the G₁/S checkpoint [99]. The effect of AUM302 may be due to reduced expression of transcription factors like c-Myc, regulating cellular metabolism and protein translation through mTOR and AKT, regulating apoptosis through BAD, and decreasing the phosphorylation and activity of the ribosomal protein S6 [100]. Our results demonstrated that 24-hour treatment with AUM302 inhibited the phosphorylation of mTOR, AKT, and S6, while the total levels of the appropriate proteins were almost unchanged (Figs 6 and 7). In contrast, TP-3654 did not have a significant effect or demonstrated minimal impact on the expression levels of these proteins. In addition, we showed that the levels of c-Myc, an effector of the PI3K/mTOR pathway, were significantly decreased upon AUM302 compared to other treatments. This observation agrees with the previous studies demonstrating the downregulation of c-Myc levels upon inhibition of PI3K/mTOR in several cancer types [101–103]. Importantly, PIM1 was shown to phosphorylate c-Myc and increase its stability [40]. Thus, the downregulation of c-Myc in our model could be due to inhibition of kinase activity of PIM and an increase in c-Myc degradation.

We further investigated the effect of AUM302 and TP-3654 on gemcitabine-resistant cells using the MIA PaCa-2 gemcitabine-resistant cell line. Treatment with TP-3654 had no significant impact on the cell viability of gemcitabine-resistant cells until a concentration of 1μM, AUM302 did have a considerable effect at 10 nM, 100 nM, and 1 μM (Figs 8 and 9). Similarly, AUM302 significantly decreased cell proliferation of gemcitabine-resistant cells compared to vehicle- and TP-3654-treated cells. This may be due to AUM302 reducing the phosphorylation and thus the activity of mTOR, AKT, and S6 and decreasing the levels of c-Myc over three-day treatment (Fig 1 and S1–S3 Figs).

This is the first in vitro study demonstrating that AUM302 is an effective inhibitor of the PIM/PI3K/mTOR pathways and decreases PDAC cell viability. Its efficacious multikinase properties make it an advantageous approach to cancer therapy compared to kinase inhibitors like TP-3654 or dual PI3K/mTOR inhibitors. Notably, the compound can overcome gemcitabine resistance in PDAC cells. AUM302 is a potent inhibitor of PDAC cell growth in vitro, and our results suggest a clinical benefit in future research. However, PDAC is characterized by great genetic intra- and inter-heterogeneity [104]. In addition, treatment outcome also depends on the interaction between tumor cells and the microenvironment. In the case of PDAC, extensive fibrosis with little vascularization limits the drug’s efficacy. Thus, to fully assess the effectiveness of AUM302, studies using models mirroring in vivo characteristics of PDAC, such as chemically-induced animal models and genetically engineered mice, patient-derived organoids, and xenografts, should be performed [105, 106].

Supporting information

S1 Fig. Densitometry analysis of western blots results of proteins regulated by PIM and PI3K/mTOR pathway in MIA PaCa-2 GemR cells treated with 10 nM, 100 nM, and 1 μM of TP-3654 or AUM302 for 24 hours.

Each experiment was performed in triplicate and the results are shown as mean ±SD (N = 3). Densitometry analysis was performed using FIJI software [61]. Statistical analysis was performed using the Student’s test followed by an analysis of the normal distribution (Tukey’s test). *p<0.05; **p<0.01; ***p<0.001.

(PDF)

Click here for additional data file.^{(111.9KB, pdf)}

S2 Fig. Densitometry analysis of western blots results of proteins regulated by PIM and PI3K/mTOR pathway in MIA PaCa-2 GemR cells treated with 10 nM, 100 nM, and 1 μM of TP-3654 or AUM302 for 48 hours.

(PDF)

Click here for additional data file.^{(110.2KB, pdf)}

S3 Fig. Densitometry analysis of western blots results of proteins regulated by PIM and PI3K/mTOR pathway in MIA PaCa-2 GemR cells treated with 10 nM, 100 nM, and 1 μM of TP-3654 or AUM302 for 72 hours.

(PDF)

Click here for additional data file.^{(101.9KB, pdf)}

S1 Raw images

(PDF)

Click here for additional data file.^{(7.5MB, pdf)}

Acknowledgments

We would like to thank Research Flow Cytometry Core in the Department of Pathology, Stony Brook University for assistance with data analysis.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

The work was supported by a grant from the National Institutes of Health awarded to ABB (DK124342). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0294065.r001

Decision Letter 0

Wagdy Mohamed Eldehna

18 Jun 2023

PONE-D-23-13615AUM302, a novel triple kinase PIM/PI3K/mTOR inhibitor, is a potent pancreatic cancer growth inhibitorPLOS ONE

Dear Dr. Bialkowska,

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #3: Yes

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Reviewer #2: Yes

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Reviewer #1: Bialkowska et al compare the effect in viablity, proliferation, cell cycle and biomarker modulation of a PIM-i with a triple inhibitor PIm/PI3K/mTOR in five pancreatic cell lines. Moreover, they compare their activity in one cell line resistant to gemcitabine. When they study the effect on biomarkers they treat the cells for 72h, a time point in which AUM-302 usually has a strong effect in viability or proliferation, so this data are not valid.

Major revision:

The authors should also include a PI3K/mTOR inhibitor to compare with and really demonstrate that the triple inhibitor is more potent

Minor revisions

1. Correct table 1. When a cell line is resistant to a drug de GI50 should be higher than the highest concentration tested

2. In order to be able to take any conclusions regarding the effect in biomarker modulation, on one hand the authors should provide a quantification of the bands and in the case of phosphorylation take into account the total levels of each corresponding protein.

Moreover, 72h is a very long time treatment to see effect in phosphorylation as compensatory mechanisms could take place.

3. In the case of resistant cell line, the highest concentration tested of AUM-302 for the evaluation of biomarkers has a very strong effect in proliferation at 72h then this results are not reliable. Shorter treatments should be included.

Reviewer #2: Running manuscript discuss a potential therapy of pancreatic cancer, UM302, as a novel triple kinase PIM/PI3K/mTOR inhibitor, is a potent pancreatic cancer. Although the point is very interesting, authors have to made major revision of their manuscript as recommended in the following points:

Methodology:

Why authors did not determine PIM1/2 inhibition to ensure the correlation with the demonstrated results and the correlation of inhibited molecular components of PIM/PI3K/m TOR

It is advisable for author to make a semiquantitative analysis of western plot bands to facilitate the interpretation of inhibited proteins

Results part:

Table one should include standard deviation of demonstrated IC50s, its title should be also more elucidated to be self-explanatory.

Quality of Figures 2-8 should be improved in their quality

Discussion:

Authors should reconstruct the discussion part to be more focused on discussing the demonstrated results and the correlation of inhibited molecular components of PIM/PI3K/m TOR.

Reviewer #3: Dr. Bialkowska and co-authors report that “AUM302, a novel triple kinase PIM/PI3K/mTOR inhibitor, is a potent pancreatic cancer growth inhibitor.” The authors should add the words in vitro to this title.

Overall, this is a well-executed and well-written study demonstrating that AUM302 inhibits the proliferation and viability of different human pancreatic cancer cell lines in culture and is capable of overcoming gemcitabine resistance in a gemcitabine-resistant MIA PaCa2 cell line.

Since the authors did not carry out in vivo studies with either intra-pancreatic or autochthonous models of PDAC, the authors should add a statement or two to the discussion section to indicate that such studies are necessary to determine whether AUM302 can be effective in vivo, and whether AUM302 is capable of altering in a beneficial manner the immune-excluding tumor microenvironment in this immune-cold cancer.

**********

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Reviewer #1: Yes: Carmen Blanco-Aparicio

Reviewer #2: No

Reviewer #3: No

**********

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PLoS One. 2023 Nov 9;18(11):e0294065. doi: 10.1371/journal.pone.0294065.r002

Author response to Decision Letter 0

19 Sep 2023

September 12th, 2023

Editor and Reviewers PLOS ONE

Re: PONE-D-23-13615

AUM302, a novel triple kinase PIM/PI3K/mTOR inhibitor, is a potent pancreatic cancer growth inhibitor

Dear Editor and Reviewers,

We want to thank the Reviewers for their invaluable comments. We also acknowledge the concerns raised by the Reviewers and now provide a revised copy of the manuscript, considering all of the Reviewers' concerns. This revised manuscript addressed all the comments and suggestions the Editor and Reviewers provided. Please see our responses to your comments below.

Reviewer 1.

Comment 1. The authors should also include a PI3K/mTOR inhibitor to compare with and really demonstrate that the triple inhibitor is more potent.

Response 1. We agree with the Reviewer comment. We performed cell viability studies in BxPC-3, Capan-2, MIA PaCa-2, PANC-1, and Hs766T using two compounds, GDC-0941 and BEZ235, both PI3K and mTOR inhibitors. We included the results in Figure 1 and Table 1 and added an appropriate statement in the Results section.

Comment 2. Correct table 1. When a cell line is resistant to a drug de GI50 should be higher than the highest concentration tested.

Response 2. We provided IC50 values in nanomolar concentrations. We performed all experiments using low concentrations of AUM-302 of 10nM and 100nM as we have observed its significant effects using these concentrations on cell proliferation and cell cycle of tested PDAC cell lines.

Comment 3. In order to be able to take any conclusions regarding the effect in biomarker modulation, on one hand the authors should provide a quantification of the bands and in the case of phosphorylation take into account the total levels of each corresponding protein. Moreover, 72h is a very long time treatment to see effect in phosphorylation as compensatory mechanisms could take place.

Response 3. We agree with the Reviewer's comment. In this revised manuscript, we included the results of the 24-hour treatment of five tested PDAC cell lines with 10nM and 100nM concentrations of TP-3654 and AUM-302 in Figure 6 and the quantification of the western blot results in Figure 7.

Comment 4. In the case of resistant cell line, the highest concentration tested of AUM-302 for the evaluation of biomarkers has a very strong effect in proliferation at 72h then this results are not reliable. Shorter treatments should be included.

Response 4. We provided western blot results from 24-, 48- and 72-hour treatment in Figure 9 and appropriate quantifications of the western blot results in Supplementary Figures 1 through 3.

Reviewer 2.

Comment 1. Why authors did not determine PIM1/2 inhibition to ensure the correlation with the demonstrated results and the correlation of inhibited molecular components of PIM/PI3K/m TOR?

Response 1. The AUM-302 compound does not impact the levels of PIM1/2 on RNA or protein levels. The compound has been designed to affect their activity. Thus, the levels of PIM1/2 are unchanged, but their activities are decreased upon AUM-302 treatment.

Comment 2. It is advisable for author to make a semiquantitative analysis of western plot bands to facilitate the interpretation of inhibited proteins.

Response 2. We agree with the Reviewer's suggestions. We performed appropriate quantifications and included them in Figure 7 and Supplementary Figures 1 through 3.

Comment 3. Table one should include standard deviation of demonstrated IC50s, its title should be also more elucidated to be self-explanatory.

Response 3. Thank you for your suggestion. We modified the title of Table 1 and provided IC50 values in nanomolar concentrations with standard deviations.

Comment 4. Quality of Figures 2-8 should be improved in their quality.

Response 4. All figures were prepared in Adobe Photoshop according to the PLOS ONE requirements with at least 300-600 dpi. All figures are submitted as .tif files. In addition, we utilized Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool recommended by PLOS ONE that tests the quality of prepared images and their compliance with the journal requirements. It could be that the journal provides low-quality figures for the primary review. We hope that the quality of the revised figures is adequate.

Comment 5. Authors should reconstruct the discussion part to be more focused on discussing the demonstrated results and the correlation of inhibited molecular components of PIM/PI3K/mTOR.

Response 5. We want to thank the Reviewer for this suggestion. We revised the Discussion section and added more information relevant to inhibiting PIM/PI3K/mTOR pathway components.

Reviewer 3.

Comment 1. The authors should add the words in vitro to this title.

Response 1. We modified the title of the manuscript to reflect its in vitro studies.

Comment 2. Since the authors did not carry out in vivo studies with either intra-pancreatic or autochthonous models of PDAC, the authors should add a statement or two to the discussion section to indicate that such studies are necessary to determine whether AUM302 can be effective in vivo, and whether AUM302 is capable of altering in a beneficial manner the immune-excluding tumor microenvironment in this immune-cold cancer.

Response 2. Thank you for this suggestion. We modified the Discussion section and added a statement emphasizing the necessity for in vivo studies to assess AUM-302 full impact on the PDAC microenvironment.

We confirm that neither the manuscript nor any parts of its content are currently under consideration or published in another journal. We changed the revised manuscript to ascertain compliance with the PLOS ONE journal's requirements.

We hope the incorporated changes will satisfy the Reviewers and Editor and render the revised manuscript suitable for publication. Thank you for being so considerate.

All authors have approved the manuscript and agree with its submission to PLOS ONE.

Please feel free to contact me if I can be of further assistance,

Sincerely Yours,

Agnieszka B. Bialkowska, PhD

Associate Professor

Renaissance School of Medicine at Stony Brook University

Department of Medicine

GI Translational Research Lab

HSC-T17 Room 090

Stony Brook, NY 11794-8176

Phone: (631) 638 2161

Email: Agnieszka.Bialkowska@stonybrookmedicine.edu

Attachment

Submitted filename: Response to the Reviewers comments.pdf

Click here for additional data file.^{(237.7KB, pdf)}

PLoS One. doi: 10.1371/journal.pone.0294065.r003

Decision Letter 1

Wagdy Mohamed Eldehna

25 Oct 2023

AUM302, a novel triple kinase PIM/PI3K/mTOR inhibitor, is a potent in vitro pancreatic cancer growth inhibitor

PONE-D-23-13615R1

Dear Dr. Bialkowska,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Wagdy Mohamed Eldehna, Ph.d

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: Authors have addressed all my comments properly. They just only need to rewrite lines 244 and 245 regarding the time treatment, as for DMSO they refer 72h and for treatment inhibitors 24h. all time points should be 24h.

Reviewer #3: Dr. Bialkowska and co-authors have addressed my concerns and the paper in my opinion presents important new information in a valid manner and is acceptable for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #1: Yes: Carmen Blanco-Aparicio

Reviewer #3: No

**********

PLoS One. doi: 10.1371/journal.pone.0294065.r004

Acceptance letter

Wagdy Mohamed Eldehna

31 Oct 2023

PONE-D-23-13615R1

AUM302, a novel triple kinase PIM/PI3K/mTOR inhibitor, is a potent in vitro pancreatic cancer growth inhibitor

Dear Dr. Bialkowska:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

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Associated Data

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Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

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PERMALINK

AUM302, a novel triple kinase PIM/PI3K/mTOR inhibitor, is a potent in vitro pancreatic cancer growth inhibitor

Komala Ingle

Joseph F LaComb

Lee M Graves

Antonio T Baines

Agnieszka B Bialkowska

Roles

Abstract

Introduction

Materials and methods

Cell lines and compounds

Cell viability assay

Cell proliferation assay

Cell cycle assay

Western blot analysis

Statistical analysis

Results

AUM302 is a potent inhibitor of pancreatic cancer cell lines growth in vitro

Fig 1. Gemcitabine, BEZ235, GDC-0941, TP-3654, and AUM302 inhibit viability of multiple pancreatic cancer cell lines.

Table 1. Experimental analysis of IC50 values of gemcitabine, BEZ235, GDC-0941, TP-3654, and AUM302 tested in BxPC-3, Capan-2, MIA PaCa-2, PANC-1, and Hs766T pancreatic cancer cell lines.

Fig 2. AUM302 inhibits the proliferation of multiple pancreatic cancer cell lines.

AUM302 alters cell cycle progression of pancreatic cancer cell lines

Fig 3. AUM302 changes the cell cycle profile of BxPC-3 and Capan-2 pancreatic cancer cell lines.

Fig 5. AUM302 changes the cell cycle profile of Hs766T pancreatic cancer cell line.

Fig 4. AUM302 changes the cell cycle profile of MIA PaCa-2 and PANC-1 pancreatic cancer cell lines.

TP-3654 and AUM302 alter the cell signaling pathways controlled by PIM kinases and PI3K/mTOR signaling pathway

Fig 6. AUM302 inhibits the cell signaling pathways regulated by PIM kinases and PI3K/mTOR pathway.

Fig 7. Densitometry analysis of western blots results of protein regulated by PIM and PI3K/mTOR pathway.

AUM302 inhibits growth of gemcitabine-resistant pancreatic cancer cells

Fig 8. AUM302 and TP-3654 decrease the cell viability of MIA PaCa-2 gemcitabine-resistant (GemR) cell line.

Fig 9. AUM302 inhibits the proliferation of MIA PaCa-2 gemcitabine-resistant (GemR) cells and activity of multiple signaling pathways.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Wagdy Mohamed Eldehna

Roles

Author response to Decision Letter 0

Decision Letter 1

Wagdy Mohamed Eldehna

Roles

Acceptance letter

Wagdy Mohamed Eldehna

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Table 1. Experimental analysis of IC₅₀ values of gemcitabine, BEZ235, GDC-0941, TP-3654, and AUM302 tested in BxPC-3, Capan-2, MIA PaCa-2, PANC-1, and Hs766T pancreatic cancer cell lines.