Combined AKT Inhibition Research Articles

Endogenous pAKT activity is associated with response to AKT inhibition alone and in combination with immune checkpoint inhibition in murine models of TNBC

Triple-negative breast cancer (TNBC) is a heterogeneous and challenging-to-treat breast cancer subtype. The clinical introduction of immune checkpoint inhibitors (ICI) for TNBC has had mixed results, and very few patients achieved a durable response. The PI3K/AKT pathway is frequently mutated in breast cancer. Given the important roles of the PI3K pathway in immune and tumor cell signaling, there is an interest in using inhibitors of this pathway to increase the response to ICI. This study sought to determine if AKT inhibition could enhance the response to ICI in murine TNBC models. We further sought to understand underlying mechanisms of response or non-response to AKT inhibition in combination with ICI. Using four murine TNBC-like cell lines and corresponding orthotopic mouse tumor models, we found that hyperactivity of the PI3K pathway, as evidenced by levels of phospho-AKT rather than PI3K pathway mutational status, was associated with response to AKT inhibition alone and in combination with ICI. Additional mutations in other growth regulatory pathways could override the response of PI3K pathway mutant tumors to AKT inhibition. Furthermore, we observed that AKT inhibition enhanced the response to ICI in an already sensitive model. However, AKT inhibition failed to convert ICI-resistant tumors, to responsive tumors. These findings suggest that analysis of both the mutational status and phospho-AKT protein levels may be beneficial in predicting which TNBC tumors will respond to AKT inhibition in combination with ICI.

Abstract PD5-08: PD5-08 Drug target activation mapping of matched triple negative primary and axillary lymph node metastases identifies and links the ER-AR-GR steroid hormone receptor axis with downstream AKT activation

Abstract Title: Drug target activation mapping of matched triple negative primary and axillary LN metastases identifies and links the ER-AR-GR steroid hormone receptor axis with downstream AKT activation Background: Triple negative breast cancer (TNBC) is comprised of heterogeneous subtypes that arise from various molecular alterations that result in TNBC having the poorest overall prognosis. There is growing interest in more sensitive ways to measure estrogen receptor (ER) and HER2 levels in the ER “negative” and HER2 “low/negative” settings, as there are patients with TNBC whose tumors are actually ER and/or HER2 “expressing/active”, and who may benefit from ER- and/or HER2-targeted therapies. Given the limitations of IHC in reliably quantitating ER and HER2 in low (0-1+) expressing tumors, we utilized highly sensitive reverse phase protein array (RPPA) to quantitate expression of these therapeutic targets along with downstream signaling activation mapping in a unique pilot set of patient-matched primary (P) and axillary lymph node (LN) metastases obtained synchronously. The quantitative expression of steroid hormone receptor expression/activation (ER, glucocorticoid receptor (GR), androgen receptor (AR)) along with their signaling architecture and dynamics were analyzed. Methods: A 12 P-LN paired tumor set was chosen for analysis (N=24). Manual macrodissection was used to enrich for tumor epithelium prior to RPPA analysis that quantitatively measured 155 proteins/phosphoprotein drug targets in key signaling pathways known to be involved in steroid hormone receptor biology and tumorigenesis. Statistical analysis using paired sample t-testing or Wilcoxon rank testing was performed (p< 0.05 uncorrected). Results: Protein pathway activation mapping and unsupervised clustering analysis revealed a subset of TNBCs from 3 of the 12 (25%) patients (N=4: 1 LN and 1 P, and one patient-matched LN and P) that was characterized by high relative levels of total ER. Interestingly, these same tumors also had amongst the highest relative levels of both total AR and activated (phosphorylated) GR compared to the rest of the 20 tumor samples. Downstream signaling analysis found significant (p< 0.05) activation/phosphorylation of AKT (S473 and/or T308) associated with the ER-AR-GR/steroid hormone activated/”high” TNBC signature compared to the 20 tumors that were steroid hormone “low”. Conclusions: Functional protein pathway activation mapping of a unique study set of synchronously obtained TNBC primary and LN metastasis revealed a steroid hormone receptor expression/activation signature of co-incidentally activated/expressed ER, GR and AR, that was also characterized by increased AKT activation (p< 0.05). This steroid hormone receptor “activated” signature was not observed to be significantly differentially expressed in LN compared to P tissues in cohort or matched pair analysis, suggesting that this signature may be present as an intrinsic event in the P, and not acquired in LN metastases. Recent data from stage I-III TNBC patients treated in the neoadjuvant setting with an AKT inhibitor1 revealed that responding TNBC patients had pre-treatment tumor biopsies that were enriched for both high AR expression and high phosphoAKT (S473 and/or T308) levels. If these observations that a subset of TNBCs are steroid hormone receptor “activated” despite being ER “negative” are confirmed in larger study sets, we hypothesize that this subgroup, identified by RPPA analysis of both P and LN metastases, could be potentially sensitive to AKT inhibition in combination with agents that target steroid hormone receptors. 1Shi Z, et al. Functional Mapping of AKT Signaling and Biomarkers of Response from the FAIRLANE Trial of Neoadjuvant Ipatasertib plus Paclitaxel for Triple-Negative Breast Cancer. Clin Cancer Res. 2022 Mar 1;28(5):993-1003 Citation Format: Julia Wulfkuhle, Page Blas, Heather Williams, Claudius Mueller, Rosa I. Gallagher, Mariaelena Pierobon, Joyce O’Shaughnessy, Emanuel F. Petricoin. PD5-08 Drug target activation mapping of matched triple negative primary and axillary lymph node metastases identifies and links the ER-AR-GR steroid hormone receptor axis with downstream AKT activation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-08.

Abstract P1-18-22: AKT inhibition in combination with endocrine therapy and a CDK4/6 inhibitor (CDK4/6i) in patients with hormone receptor positive (HR+)/HER2 negative metastatic breast cancer (MBC) and prior CDK4/6i exposure: A translational investigation

Abstract Background: The cyclin-dependent kinase 4/6 inhibitors, with endocrine therapy (ET), have become the standard of care for patients with HR+/HER2- MBC. Prior insight from tumor biopsies and preclinical analyses suggest that AKT1 activation can provoke CDK4/6i resistance, highlighting a potential therapeutic role for AKT inhibition (AKTi) in this setting. However, combinatorial inhibition can be associated with significant toxicity and identification of the optimal biological dose is often challenging. In this translational co-clinical study, we evaluated escalating doses of AKTi combination with CDK 4/6i in parallel patient-derived pre-clinical models as well as a phase 1b clinical trial. Methods: In an open-label phase Ib dose-escalation clinical trial (TAKTIC, NCT03959891), we evaluated the safety, tolerability and efficacy of escalating doses of the AKT1 inhibitor ipatasertib (ipat) in combination with palbociclib (palbo) and fulvestrant (fulv) for patients with HR+/HER2- MBC. Inclusion criteria include unresectable or metastatic disease, at least 1 prior therapy for MBC including any CDK4/6i, and up to 2 prior lines of chemotherapy for MBC (no limit on prior endocrine therapy). In addition, response to escalating doses of ipat and palbo (with fulv) were explored in vitro via an ATP-based viability assay in tumor cell lines derived from circulating tumor cells (CTC) isolated from patients with endocrine-refractory HR+ MBC. Results: In the dose-escalation portion of the phase 1b clinical trial, 23 patients received the triplet combination of ipat, palbo, and fulv (median number of prior lines = 4.3, range 1-7; 100% with prior CDK4/6i): 3 pts received ipat at 200mg + 125mg palbo, 15 pts received 300mg + 125mg palbo, and 5 pts received 400mg + 100mg palbo, all with fulv (500 mg). Among the 23 patients, 20 patients (86.9%) had disease control (4 partial response and 16 stable disease) as the best response, per RECIST. Grade 3/4 toxicities included neutropenia (n=20), lymphopenia (n=3), diarrhea (n=3), thrombocytopenia (n=2), transaminitis (n=2), and rash (n=2). Two DLTs were observed in the 300mg ipat + 125mg palbo cohort (grade 4 neutropenia ≥ 7 days), but none at 400mg + 100mg palbo. The combination of ipat and palbo demonstrated an additive effect in vitro, with increased sensitivity to lower doses of palbo in the presence of ipat. Based on the totality of data, 400mg ipat + 100mg palbo + fulv 500 mg was selected as the recommended phase II dose (RP2D) in the post-CDK4/6i setting. Conclusions: The triplet combination of endocrine therapy with AKTi and lower dose CDK4/6i appears to be well tolerated in heavily pre-treated pts, with preliminary evidence of clinical activity. Further study is needed to evaluate biomarkers associated with higher AKTi benefit in order to guide rational development of combination therapy for patients with HR+/HER2- MBC in the post-CDK4/6i setting. Overall, this translational study demonstrates how insight into the molecular mechanisms of CDK4/6i resistance and combinatorial modeling can be leveraged to develop actionable therapeutic regimens for patients with MBC. Citation Format: Seth A. Wander, Douglas S. Micalizzi, Taronish Dubash, Dejan Juric, Laura M. Spring, Neelima Vidula, Jennifer Keenan, Maureen Beeler, Elene Viscosi, Dante Che, Elizabeth L. Fisher, Rachel A. Hepp, Beverly Moy, Steven J. Isakoff, Leif W. Ellisen, Jeffrey G. Supko, Shyamala Maheswaran, Daniel A. Haber, Aditya Bardia. AKT inhibition in combination with endocrine therapy and a CDK4/6 inhibitor (CDK4/6i) in patients with hormone receptor positive (HR+)/HER2 negative metastatic breast cancer (MBC) and prior CDK4/6i exposure: A translational investigation [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-18-22.

Combined Inhibition of AKT and KIT Restores Expression of Programmed Cell Death 4 (PDCD4) in Gastrointestinal Stromal Tumor.

Simple SummaryImatinib mesylate (IM, Gleevec) has remained first-line therapy for most gastrointestinal stromal tumor (GIST) patients since 2002. During this time, three additional inhibitors have been approved for treatment of IM-refractory tumors; however, disease stabilization for these agents is measured in months. Once all approved lines of therapy are ineffective, patients with advanced GIST are left without treatment options. Therefore, preventing or delaying development of acquired resistance to IM could have clinical benefits. Activation of the PI3K/AKT signaling pathway has been associated with resistance to IM in GIST. We evaluated MK-4440, a novel AKT inhibitor, in combination with IM in GIST cells and mouse models. Our studies demonstrate superior activity of MK-4440/IM combination in a panel of GIST cell lines caused by cell cycle arrest and elevated PDCD4 expression leading to increased cell death. Furthermore, dual inhibition of KIT and AKT provided impressive disease stabilization in IM-sensitive GIST growing in mice.The majority of gastrointestinal stromal tumor (GIST) patients develop resistance to the first-line KIT inhibitor, imatinib mesylate (IM), through acquisition of secondary mutations in KIT or bypass signaling pathway activation. In addition to KIT, AKT is a relevant target for inhibition, since the PI3K/AKT pathway is crucial for IM-resistant GIST survival. We evaluated the activity of a novel pan-AKT inhibitor, MK-4440 (formerly ARQ 751), as monotherapy and in combination with IM in GIST cell lines and preclinical models with varying IM sensitivities. Dual inhibition of KIT and AKT demonstrated synergistic effects in IM-sensitive and -resistant GIST cell lines. Proteomic analyses revealed upregulation of the tumor suppressor, PDCD4, in combination treated cells. Enhanced PDCD4 expression correlated to increased cell death. In vivo studies revealed superior efficacy of MK-4440/IM combination in an IM-sensitive preclinical model of GIST compared with either single agent. The combination demonstrated limited efficacy in two IM-resistant models, including a GIST patient-derived xenograft model possessing an exon 9 KIT mutation. These studies provide strong rationale for further use of AKT inhibition in combination with IM in primary GIST; however, alternative agents will need to be tested in combination with AKT inhibition in the resistant setting.

Pharmacologic inhibition of Akt in combination with chemotherapeutic agents effectively induces apoptosis in ovarian and endometrial cancer cell lines.

The PI3K/Akt signaling pathway, the most frequently altered signaling system in human cancer, is a crucial inducer of dysregulated proliferation and neoplastic processes; however, few therapeutic strategies using PI3K/Akt inhibitors singly have been shown to be effective. The purpose of this paper was to underline the potential benefit of pharmacological modulation of the PI3K/Akt pathway when combined with specific chemotherapeutic regimens. We have studied the ability of NVP‐BEZ235 (PI3K/mTOR inhibitor) and AZD5363 (Akt inhibitor) in the sensitization of cancer cells to cisplatin and doxorubicin. Our results show that NVP‐BEZ235 sensitizes cells preferentially to cisplatin while AZD5363 sensitizes cells to doxorubicin. At equal concentrations (5 μm), both inhibitors reduce ribosomal protein S6 phosphorylation, but AZD5363 is more effective in reducing GSK3β phosphorylation as well as S6 phosphorylation. Additionally, AZD5363 is capable of inducing FOXO1 and p53 nuclear localization and reduces BAD phosphorylation, which is generally increased by cisplatin and doxorubicin. Finally, the combination of AZD5363 and doxorubicin induces apoptosis in cells and robustly reduces cell ability to clonally replicate, which underlines a potential cooperative effect of the studied compounds.

Abstract 669: Combined inhibition of AKT & TBK1 for the treatment of breast cancer

Abstract Breast cancer (BC) is the most frequently diagnosed malignancy and the second leading cause of cancer mortality in Western women. In ~80% of BC cases, the PI3K-AKT pathway is aberrantly activated, due to the alterations in genes encoding the pathway components, such as Ras, Her2, PTEN, PIK3C and AKT. This pathway regulates multiple cellular processes to promote BC development, growth, metastasis, and drug resistance. Consequently, over 100 clinical trials are currently underway worldwide to evaluate the therapeutic efficacy of PI3K and AKT inhibitors in BC; however, initial data reported so far revealed that inhibition of this pathway is either not effective or often results in development of resistance and relapse of the disease. Thus, identification of additional targets and therapeutic combinations are urgently needed. We previously mapped TRAF2 phosphorylation sites (Ser-11 and Ser-55), identified PKC and TBK1 are the relevant kinases, and reported that TRAF2 phosphorylation enhances NF-κB activation to promote cancer cell survival under conditions of cellular stresses. Recently, we discovered that inhibition of AKT leads to increased phosphorylation of TRAF2 at Ser-11 by TBK1 in 4 out of 6 commonly used BC cell lines. TBK1 (aka T2K for TRAF2-associated kinase) and its close homologue IKKϵ are serine/threonine kinases overexpressed in ~70% of BC and play critical roles in BC cell survival. Interestingly, all 4 BC cell lines that exhibited enhanced TRAF2 phosphorylation were completely resistant to AKT inhibition-induced apoptosis in vitro, and combined inhibition of both AKT and TBK1/IKKϵ synergistically induced apoptosis in these BC lines. Importantly, combined inhibition of AKT and TBK1/IKKϵ also significantly suppresses xenograft BC tumor growth in vivo. Mechanistically, AKT did not directly inhibit TBK1, IKKϵ or TRAF2; it seems that an unknown kinase suppressed by AKT in cancer cells in normal culture conditions enhances TBK1-mediated TRAF2 Ser-11 phosphorylation in BC cells upon AKT inhibition. Nevertheless, our data suggest that inhibition of the PI3K-AKT pathway derepresses the pro-survival TBK1-TRAF2 pathway, which in turn confers resistance to PI3K-AKT inhibition in BC, and that combined inhibition of AKT and TBK1 effectively suppresses BC tumor growth. These findings also provide strong rationales for PDX and clinical investigation of combined AKT and TBK1 inhibition for the treatment of human BCs. Citation Format: Hasem Habelhah, Laiqun Zhnag. Combined inhibition of AKT & TBK1 for the treatment of breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 669.

Abstract 6383: HTBPI inhibits liver tumorigenesis by combined inhibition of Akt

Abstract Akt, a crucial protein involved in a variety of signaling pathways in cancer, acts as an important regulator of cell survival in hepatocellular carcinoma (HCC), and provides curative option for the related drug development. We have found an active phenanthroindolizidine alkaloid, (13aR,14R)-9,11,12,13,13a,14-hexahydro-3,6,7- trimethoxydibenzo[f,h]pyrrolo[1,2-b]isoquinolin-14-ol (HTBPI), which is a promising Akt inhibitor effective in the suppression of HCC cells proliferation through stimulating apoptotic and autophagic capability in vivo and in vitro. Treatment of HTBPI combined with a classical autophagy-lysosomal inhibitor (bafilomycin A1), could enhance stimulation effects of apoptosis on HCC cell lines. In addition, we confirmed that HTBPI, targeting Akt, occupied the kinase binding domain (Thr 308) of Akt to inactivate its function by CETSA and DARTS assay. Moreover, HTBPI showed a potential therapeutic effect in patient-derived xenograft (PDX) tumors without observed toxicity. Furthermore, high p-Akt (Thr 308) expression was collated with liver tumor formation and poor survival in HCC patients. In conclusions, HTBPI impeded HCC progress through regulation of apoptosis and autophagy machinery via interaction with p-Akt (Thr 308). This may provide potential molecular candidate by inhibiting Akt for the therapy of HCC patients. Citation Format: Haiyang Yu, Hongwei Liu, Qian Chen, Xu Pang, Shuangshuang Yin, Kailong Wang, Rui Wang, Tao Wang, Yuling Qiu. HTBPI inhibits liver tumorigenesis by combined inhibition of Akt [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6383.

SCD1 Confers Temozolomide Resistance to Human Glioma Cells via the Akt/GSK3β/β-Catenin Signaling Axis.

Resistance to temozolomide (TMZ), the standard chemotherapy agent for glioblastoma (GBM), poses a major clinical challenge to GBM prognosis. Understanding the mechanisms of TMZ resistance can help to identify novel drug targets and more effective therapies. Recent studies suggest that bioenergetic alterations of cancer cells play important roles in drug resistance. In our study, the altered metabolism of cancer cells was observed using a metabolic PCR array. We found that stearoyl-coenzyme A desaturase 1 (SCD1), a key rate-limiting enzyme for synthesis of monounsaturated fatty acids, was significantly upregulated in TMZ-resistant GBM cells compared to their parental counterparts. Overexpression of SCD1 promoted resistance to TMZ in parental GBM cells, whereas SCD1 downregulation by siRNA could re-sensitize TMZ-resistant cells in vitro. Combinational treatment of TMZ and an SCD1-specific inhibitor showed a combined inhibitory effect on TMZ-resistant glioma cells. We also observed that overexpression of SCD1 promoted Akt/GSK3β/β-catenin signaling, while silencing of SCD1 inhibited the signaling. The combination of an Akt activator with exogenous SCD1 or the combined inhibition of Akt and enforced expression of SCD1 resulted in the most significant changes of Akt signaling. Functionally, significantly lower viability and mobility rates were observed in TMZ-resistant cells when treated with Akt inhibitors and an SCD1 inhibitor simultaneously compared to when treated individually. In conclusion, our study identified SCD1 along with its functional pathway as a novel target in the development of TMZ resistance. SCD1 inhibition used alone or in combination with Akt inhibition could effectively overcome TMZ resistance in gliomas.

Combined inhibition of AKT and HSF1 suppresses breast cancer stem cells and tumor growth.

Breast cancer is the most common cancer in women and the second leading cause of cancer deaths in women. Over 90% of breast cancer deaths are attributable to metastasis. Our lab has recently reported that AKT activates heat shock factor 1 (HSF1), leading to epithelial-to-mesenchymal transition in HER2-positive breast cancer. However, it is unknown whether the AKT-HSF1 pathway plays an important role in other breast cancer subtypes, breast cancer stem cells, or breast cancer growth and metastasis. Herein, we showed AKT and HSF1 to be frequently co-activated in breast cancer cell lines and specimens across different subtypes. Activated AKT (S473) and HSF1 (S326) are strongly associated with shortened time to metastasis. Inhibition of the AKT-HSF1 signaling axis using small molecule inhibitors, HSF1 knockdown or the dominant-negative HSF1 mutant (S326A) reduced the growth of metastatic breast cancer cells and breast cancer stem cells. The combination of small molecule inhibitors targeting AKT (MK-2206) and HSF1 (KRIBB11) resulted in synergistic killing of breast cancer cells and breast cancer stem cells across different molecular subtypes. Using an orthotopic xenograft mouse model, we found that combined targeting of AKT and HSF1 to significantly reduce tumor growth, induce tumor apoptosis, delay time to metastasis, and prolong host survival. Taken together, our results indicate AKT-HSF1 signaling mediates breast cancer stem cells self-renewal, tumor growth and metastasis, and that dual targeting of AKT and HSF1 resulted in synergistic suppression of breast cancer progression thereby supporting future testing of AKT-HSF1 combination therapy for breast cancer patients.

Anti-proliferative and anti-secretory effects of everolimus on human pancreatic neuroendocrine tumors primary cultures: is there any benefit from combination with somatostatin analogs?

Therapeutic management of gastroenteropancreatic neuroendocrine tumors (GEP-NETs) is challenging. The mammalian target of rapamycin (mTOR) inhibitor everolimus recently obtained approval from the Food and Drug Administration for the treatment of patients with advanced pancreatic neuroendocrine tumors (pNETs). Despite its promising antitumor efficacy observed in cell lines, clinical benefit for patients is unsatisfactory. The limited therapeutic potential of everolimus in cancer cells has been attributed to Akt activation due to feedback loops relief following mTOR inhibition. Combined inhibition of Akt might then improve everolimus antitumoral effect. In this regard, the somatostatin analog (SSA) octreotide has been shown to repress the PI3K/Akt pathway in some tumor cell lines. Moreover, SSAs are well tolerated and routinely used to reduce symptoms caused by peptide release in patients carrying functional GEP-NETs. We have recently established and characterized primary cultures of human pNETs and demonstrated the anti-proliferative effects of both octreotide and pasireotide. In this study, we aim at determining the antitumor efficacy of everolimus alone or in combination with the SSAs octreotide and pasireotide in primary cultures of pNETs. Everolimus reduced both Chromogranin A secretion and cell viability and upregulated Akt activity in single treatment. Its anti-proliferative and anti-secretory efficacy was not improved combined with the SSAs. Both SSAs did not overcome everolimus-induced Akt upregulation. Furthermore, caspase-dependent apoptosis induced by SSAs was lost in combined treatments. These molecular events provide the first evidence supporting the lack of marked benefit in patients co-treated with everolimus and SSA.

The allosteric AKT inhibitor MK2206 shows a synergistic interaction with chemotherapy and radiotherapy in glioblastoma spheroid cultures

BackgroundGlioblastoma multiforme (GBM) is the most common, invasive and deadly primary type of malignant brain tumor. The Phosphatidylinositol-3-Kinase/AKT (PI3K/AKT) pathway is highly active in GBM and has been associated with increased survival and resistance to therapy. The aim of this study is to investigate the effects of AKT inhibition in combination with the current standard of care which consists of irradiation and temozolomide (TMZ) on human malignant glioma cells growing adherent and as multicellular spheroids in vitro.MethodsThe effects of the allosteric inhibitor MK2206 combined with irradiation and TMZ were assessed on glioma cells growing adherent and as multicellular 3D spheroids. The interaction was studied on proliferation, clonogenic cell survival, cell invasion, −migration and on expression of key proteins in the PI3K-AKT pathway by western blot.ResultsA differential effect was found at low- (1 μM) and high dose (10 μM) MK2206. At 1 μM, the inhibitor reduced phosphorylation of Thr308 and Ser473 residues of AKT in both adherent cells and spheroids. Low dose MK2206 delayed spheroid growth and sensitized spheroids to both irradiation and TMZ in a synergistic way (Combination index <0.35). In contrast, neither low nor high dose MK2206 did enhance therapy sensitivity in adherent growing cells. Effective inhibition of invasion and migration was observed only at higher doses of MK2206 (>5 μM).ConclusionsThe data show that a 3D spheroid model show different sensitivity to irradiation when combined with AKT inhibition. Thereby we show that MK2206 has potential synergistic efficacy to the current standard of care for glioma patients.

Phospho-Akt overexpression is prognostic and can be used to tailor the synergistic interaction of Akt inhibitors with gemcitabine in pancreatic cancer

BackgroundThere is increasing evidence of a constitutive activation of Akt in pancreatic ductal adenocarcinoma (PDAC), associated with poor prognosis and chemoresistance. Therefore, we evaluated the expression of phospho-Akt in PDAC tissues and cells, and investigated molecular mechanisms influencing the therapeutic potential of Akt inhibition in combination with gemcitabine.MethodsPhospho-Akt expression was evaluated by immunohistochemistry in tissue microarrays (TMAs) with specimens tissue from radically-resected patients (n = 100). Data were analyzed by Fisher and log-rank test. In vitro studies were performed in 14 PDAC cells, including seven primary cultures, characterized for their Akt1 mRNA and phospho-Akt/Akt levels by quantitative-RT-PCR and immunocytochemistry. Growth inhibitory effects of Akt inhibitors and gemcitabine were evaluated by SRB assay, whereas modulation of Akt and phospho-Akt was investigated by Western blotting and ELISA. Cell cycle perturbation, apoptosis-induction, and anti-migratory behaviors were studied by flow cytometry, AnnexinV, membrane potential, and migration assay, while pharmacological interaction with gemcitabine was determined with combination index (CI) method.ResultsImmunohistochemistry of TMAs revealed a correlation between phospho-Akt expression and worse outcome, particularly in patients with the highest phospho-Akt levels, who had significantly shorter overall and progression-free-survival. Similar expression levels were detected in LPC028 primary cells, while LPC006 were characterized by low phospho-Akt. Remarkably, Akt inhibitors reduced cancer cell growth in monolayers and spheroids and synergistically enhanced the antiproliferative activity of gemcitabine in LPC028, while this combination was antagonistic in LPC006 cells. The synergistic effect was paralleled by a reduced expression of ribonucleotide reductase, potentially facilitating gemcitabine cytotoxicity. Inhibition of Akt decreased cell migration and invasion, which was additionally reduced by the combination with gemcitabine. This combination significantly increased apoptosis, associated with induction of caspase-3/6/8/9, PARP and BAD, and inhibition of Bcl-2 and NF-kB in LPC028, but not in LPC006 cells. However, targeting the key glucose transporter Glut1 resulted in similar apoptosis induction in LPC006 cells.ConclusionsThese data support the analysis of phospho-Akt expression as both a prognostic and a predictive biomarker, for the rational development of new combination therapies targeting the Akt pathway in PDAC. Finally, inhibition of Glut1 might overcome resistance to these therapies and warrants further studies.

Jagged 1 is a major Notch ligand along cholangiocarcinoma development in mice and humans.

Intrahepatic cholangiocarcinoma (ICC) is a rare yet deadly malignancy with limited treatment options. Activation of the Notch signalling cascade has been implicated in cholangiocarcinogenesis. However, while several studies focused on the Notch receptors required for ICC development, little is known about the upstream inducers responsible for their activation. Here, we show that the Jagged 1 (Jag1) ligand is almost ubiquitously upregulated in human ICC samples when compared with corresponding non-tumorous counterparts. Furthermore, we found that while overexpression of Jag1 alone does not lead to liver tumour development, overexpression of Jag1 synergizes with activated AKT signalling to promote liver carcinogenesis in AKT/Jag1 mice. Histologically, tumours consisted exclusively of ICC, with hepatocellular tumours not occurring in AKT/Jag1 mice. Furthermore, tumours from AKT/Jag1 mice exhibited extensive desmoplastic reaction, an important feature of human ICC. At the molecular level, we found that both AKT/mTOR and Notch cascades are activated in AKT/Jag1 ICC tissues, and that the Notch signalling is necessary for ICC development in AKT/Jag1 mice. In human ICC cell lines, silencing of Jag1 via specific small interfering RNA reduces proliferation and increases apoptosis. Finally, combined inhibition of AKT and Notch pathways is highly detrimental for the in vitro growth of ICC cell lines. In summary, our study demonstrates that Jag1 is an important upstream inducer of the Notch signalling in human and mouse ICC. Targeting Jag1 might represent a novel therapeutic strategy for the treatment of this deadly disease.

Heat Stress-Induced PI3K/mTORC2-Dependent AKT Signaling Is a Central Mediator of Hepatocellular Carcinoma Survival to Thermal Ablation Induced Heat Stress.

Thermal ablative therapies are important treatment options in the multidisciplinary care of patients with hepatocellular carcinoma (HCC), but lesions larger than 2–3 cm are plagued with high local recurrence rates and overall survival of these patients remains poor. Currently no adjuvant therapies exist to prevent local HCC recurrence in patients undergoing thermal ablation. The molecular mechanisms mediating HCC resistance to thermal ablation induced heat stress and local recurrence remain unclear. Here we demonstrate that the HCC cells with a poor prognostic hepatic stem cell subtype (Subtype HS) are more resistant to heat stress than HCC cells with a better prognostic hepatocyte subtype (Subtype HC). Moreover, sublethal heat stress rapidly induces phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dependent-protein kinase B (AKT) survival signaling in HCC cells in vitro and at the tumor ablation margin in vivo. Conversely, inhibition of PI3K/mTOR complex 2 (mTORC2)-dependent AKT phosphorylation or direct inhibition of AKT function both enhance HCC cell killing and decrease HCC cell survival to sublethal heat stress in both poor and better prognostic HCC subtypes while mTOR complex 1 (mTORC1)-inhibition has no impact. Finally, we showed that AKT isoforms 1, 2 and 3 are differentially upregulated in primary human HCCs and that overexpression of AKT correlates with worse tumor biology and pathologic features (AKT3) and prognosis (AKT1). Together these findings define a novel molecular mechanism whereby heat stress induces PI3K/mTORC2-dependent AKT survival signaling in HCC cells and provide a mechanistic rationale for adjuvant AKT inhibition in combination with thermal ablation as a strategy to enhance HCC cell killing and prevent local recurrence, particularly at the ablation margin.

Differential regulation of mTOR signaling determines sensitivity to AKT inhibition in diffuse large B cell lymphoma.

Agents that target components of the PI3K/AKT/mTOR pathway are under investigation for the treatment of diffuse large B cell lymphoma (DLBCL). Given the highly heterogeneous nature of DLBCL, it is not clear whether all subtypes of DLBCL will be susceptible to PI3K pathway inhibition, or which kinase within this pathway is the most favorable target. Pharmacological profiling of a panel of DLBCL cell lines revealed a subset of DLBCL that was resistant to AKT inhibition. Strikingly, sensitivity to AKT inhibitors correlated with the ability of these inhibitors to block phosphorylation of S6K1 and ribosomal protein S6. Cell lines resistant to AKT inhibition activated S6K1 independent of AKT either through upregulation of PIM2 or through activation by B cell receptor (BCR) signaling components. Finally, combined inhibition of AKT and BTK, PIM2, or S6K1 proved to be an effective strategy to overcome resistance to AKT inhibition in DLBCL.

Abstract 809: BRAF wild-type melanoma survival hinges upon AKT activity and adaptive autophagic reprogramming in response to chemotherapy

Abstract Strategies for patients whose melanomas are BRAF wild-type are completely lacking. We here report on the mechanisms underlying the long-term responses (&amp;gt;12 months) of 2 BRAF wild-type (WT) melanoma patients to the AKT inhibitor MK-2206 in combination with paclitaxel and carboplatin. A panel of 7 BRAF/NRAS-WT cell lines was leveraged to investigate strategies to overcome chemoresistance. Electron microscopy, western blotting and flow cytometry assays were integrated to simultaneously follow fluctuations in autophagic flux, reactive oxygen species levels and cytotoxicity caused from chronic treatment of cells with chemotherapy, MK-2206, lysomotropic agents and/or anti-oxidants. MK-2206 potently inhibited phospho-AKT signaling, but did not impact melanoma growth or survival. The combination of MK-2206 with paclitaxel and carboplatin induced little apoptosis following 3-day treatment, but led to cytotoxicity in long-term colony formation and 3D spheroid assays. Mechanistically the combination was noted to induce autophagy and disrupt reactive oxygen species homeostasis, as shown by increased LC3-II expression, the formation of LC3 punctae, acridine orange protonation, the accumulation of autophagosomes and increased ROS detection using DHR-123. At earlier time points, autophagy was protective with cytotoxicity enhanced following chloroquine and bafilomycin treatment as well as the increased cytotoxic effects seen in ATG5-/- MEFs. Although prolonged MK-2206/chemotherapy treatment (6-9 days) led to high levels of autophagy induction and a switch to caspase-dependent apoptosis, drug resistant clones still eventually emerged. The importance of autophagy for the escape process was suggested by the lack of ATG5-/- MEF colonies seen following combination therapy treatment. Autophagy inhibition enhanced the cell death response through increased generation of ROS and could be reversed by anti-oxidants. In summary, we demonstrate for the first time that AKT inhibition in combination with chemotherapy may have clinical activity in BRAF WT melanoma. We further show that the administration of an autophagy inhibitor may prevent resistance from emerging to this drug combination. Citation Format: Vito W. Rebecca, Renato Massaro, Inna Fedorenko, Geoffrey T. Gibney, Vernon K. Sondak, Ravi K. Amaravadi, Shengkan Jin, Silvya S. Maria-Engler, Ragini Kudchadkar, Keiran S. M. Smalley. BRAF wild-type melanoma survival hinges upon AKT activity and adaptive autophagic reprogramming in response to chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 809. doi:10.1158/1538-7445.AM2014-809

FGFR2 is amplified in the NCI-H716 colorectal cancer cell line and is required for growth and survival.

Aberrant kinase activation resulting from mutation, amplification, or translocation can drive growth and survival in a subset of human cancer. FGFR2 is amplified in breast and gastric cancer, and we report here the first characterization of FGFR2 gene amplification in colorectal cancer in the NCI-H716 colorectal cancer cell line. FGFR2 is highly expressed and activated in NCI-H716 cells, and FGFR selective small molecule inhibitors or FGFR2 shRNA strongly inhibited cell viability in vitro, indicating “addiction” of NCI-H716 cells to FGFR2. NCI-H716 growth in a xenograft model was also inhibited by an FGFR small molecule inhibitor. FGFR2 was required for activation of multiple downstream signaling proteins including AKT, ERK, S6RP and NFKB. Inhibition of downstream kinases such as AKT or ERK alone had modest effects on proliferation, whereas combined inhibition of AKT and ERK signaling resulted in a loss of viability similar to FGFR2 inhibition. We identified elevated FGFR2 expression in a small subset of primary colorectal cancer, however FGFR2 amplification was not observed. Although FGFR2 amplification is not common in primary colon cancer or lymph node and liver metastases, other subsets of colorectal cancer such as ascites, from which the NCI-H716 cell line was derived, have yet to be tested. These results suggest that emerging FGFR inhibitor therapeutics may have efficacy in a subset of colon cancer driven by FGFR2 amplification.

Inhibition of autophagy enhances the effects of the AKT inhibitor MK‐2206 when combined with paclitaxel and carboplatin in BRAF wild‐type melanoma

This study investigates the mechanism of action behind the long-term responses (12-16 months) of two BRAF WT melanoma patients to the AKT inhibitor MK-2206 in combination with paclitaxel and carboplatin. Although single agent MK-2206 inhibited phospho-AKT signaling, it did not impact in vitro melanoma growth or survival. The combination of MK-2206 with paclitaxel and carboplatin was cytotoxic in long-term colony formation and 3D spheroid assays, and induced autophagy. Autophagy was initially protective with autophagy inhibitors and deletion of ATG5 found to enhance cytotoxicity. Although prolonged autophagy induction (>6 days) led to caspase-dependent apoptosis, drug resistant clones still emerged. Autophagy inhibition enhanced the cell death response through reactive oxygen species and could be reversed by anti-oxidants. We demonstrate for the first time that AKT inhibition in combination with chemotherapy may have clinical activity in BRAF WT melanoma and show that an autophagy inhibitor may prevent resistance to these drugs.

Increasing the therapeutic efficacy of docetaxel for cutaneous squamous cell carcinoma through the combined inhibition of phosphatidylinositol 3-kinase/AKT signalling and autophagy

Early-stage cutaneous squamous cell carcinoma (cSCC) has a favourable prognosis. Metastatic disease is probably associated with chemoresistance mediated through the activation of pro-survival phosphatidylinositol 3-kinase/AKT signalling. Inhibition of activated AKT partially increases chemosensitivity but induces autophagy, the principal lysosomal mechanism for the bulk degradation and recycling of proteins and damaged organelles. The aim of the current study was to test the hypothesis that combined inhibition of AKT signalling and autophagy by the lysosomal inhibitor chloroquine increases the susceptibility to docetaxel-induced apoptosis of cSCC cells isolated from a lymph-node metastasis. Combined AKT inhibition and chloroquine treatment of MET 4 cSCC cells resulted in significantly enhanced inhibition of cell viability and apoptosis induced by clinically achievable concentrations of docetaxel (P<0.001). Inhibition of both autophagy and AKT thus represents an effective and viable therapeutic strategy to increase the cytotoxicity of docetaxel for the treatment of advanced cSCC.

Molecular Imaging Reveals a Role for AKT in Resistance to Cisplatin for Ovarian Endometrioid Adenocarcinoma

Ovarian cancer is the fifth leading cause of cancer-related deaths among American women. Platinum-based chemotherapy, such as cisplatin, represents the standard-of-care for ovarian cancer. However, toxicity and acquired resistance to cisplatin have proven challenging in the treatment of patients with ovarian cancer. Using a genetically engineered mouse model of ovarian endometrioid adenocarcinoma (OEA) in combination with molecular-imaging technologies, we studied the activation of the AKT serine/threonine kinase in response to long-term cisplatin therapy. Treatment of cells in culture and tumor-bearing animals with cisplatin resulted in activation of AKT, a key mediator of cell survival. On the basis of these results, we investigated the therapeutic use of AKT inhibition in combination with cisplatin, which resulted in enhanced and prolonged induction of apoptosis and in significantly improved tumor control as compared with either agent alone. These results provide an impetus for clinical trials using combination therapy. To facilitate these trials, we also show the use of diffusion-weighted MRI as an imaging biomarker for evaluation of therapeutic efficacy in OEA.