Therapy In Cell Lines Research Articles

AB029. Mitochondrial dysfunction and impaired growth of glioblastoma cell lines caused by antimicrobial agents inducing ferroptosis.

Glioblastoma, a malignant tumor, has no curative treatment. Mitochondria have been reported to be involved in tumorigenesis and drug resistance in various cancers. Recently, mitochondria have been considered a potential target for treating glioblastoma. We are developing a new treatment for glioblastoma targeting mitochondria. The microenvironment of glioblastoma is reported to be a low-nutrition and hypoxic condition. We focused our research on how mitochondria work under tumor circumstances. In this research, we used cell lines including U87, LN229, U373, T98G, and two patient-derived stem-like cells. First, we investigated the efficacy of mitochondria-targeted glioblastoma therapy in cell lines under glucose-starved conditions and the mechanism of cell death caused by mitochondria-targeted therapy. Second, we developed a treatment effective for cells with more glucose, because we believe that there is some lesion with higher glucose concentration in the tumor considering the heterogeneity of the tumor. We discovered that under glucose-starved conditions, mitochondria-related proteins and oxidative phosphorylation activity increased in glioblastoma cells, and in this condition, glioblastoma cells strongly depended on mitochondria. Administration of agents causing mitochondrial dysfunction including chloramphenicol was very effective and it led to cell death. The mechanism of cell death was not apoptosis but ferroptosis which is a recently discovered type of cell death caused by iron accumulation. In normoglycemic conditions, the effect of chloramphenicol was poor for the short term, however, for the long term, it was effective and caused cell death. Although both chloramphenicol alone and combined treatment using 2-DG (glycolysis inhibitor) and chloramphenicol had poor effects on cells in glucose-sufficient conditions, combined treatment was very effective for the short term under normal glucose conditions. These results suggest the importance of controlling glucose concentration. The effect of combined treatment was also confirmed in hypoxic conditions and for patient-derived neurosphere cells. And the mechanism of cell death was ferroptosis, too. Our mitochondria-targeted treatment was effective for cells with both glucose-starved conditions and normoglycemic conditions. It is necessary to investigate its effectiveness in vivo in the future. However, chloramphenicol has already been used, and therefore, this treatment can be used for humans safely. This discovery is expected to make a major contribution to the development of treatments for glioblastomas.

A key role of the WEE1-CDK1 axis in mediating TKI-therapy resistance in FLT3-ITD positive acute myeloid leukemia patients

The insertion site of the internal tandem duplications (ITDs) in the FLT3 gene affects the sensitivity to tyrosine kinase inhibitors (TKIs) therapy in acute myeloid leukemia (AML). Patients with the ITD in the tyrosine kinase domain lack effective therapeutic options. Here, to identify genotype-driven strategies increasing the TKI therapy efficacy, we developed SignalingProfiler, a strategy supporting the integration of high-sensitive mass spectrometry-based (phospho)proteomics, RNA sequencing datasets with literature-derived signaling networks. The approach generated FLT3-ITD genotype-specific predictive models and revealed a conserved role of the WEE1-CDK1 axis in TKIs resistance. Remarkably, pharmacological inhibition of the WEE1 kinase synergizes and strengthens the pro-apoptotic effect of TKIs therapy in cell lines and patient-derived primary blasts. Finally, we propose a new molecular mechanism of TKIs resistance in AML and suggest the combination of WEE1 inhibitor and TKI as a therapeutic option to improve patients clinical outcome.

CD58 Could be As a Leukemic Marker in Relapsed/Refractory B-ALL Patients after Multiline Therapies

It has been verified that CD58, a member of the immunoglobulin superfamily, is expressed in B-cell lymphoblastic leukemia (B-ALL) at a higher level and is a marker of minimal residual disease in B-ALL. A study reported that CD58 was expressed in 99.4% of B-ALL cases at diagnosis. Nevertheless, little is known regarding CD58 expression in relapsed/refractory (r/r) B-ALL patients having received multiline therapies especially chimeric antigen receptor (CAR) T-cells. Although CD19-specific CAR-T therapy resulted in high complete remission (CR) rates, some patients relapsed after CAR-T and part of them showed CD19 loss, under this circumstance, additional B-cell antigen specifically expressed on the B-ALL cells could help us to identify the leukemia cells. In this study, we investigated the CD58 expression in r/r B-ALL patients before CAR-T, and the influence of CAR-T cells on CD58 antigen of lymphoblasts in patients failed or relapsed after CAR-T. CD58 on leukemic cells of r/r B-ALL patients has been routinely detected by flow cytometry in our center. Data of r/r B-ALL patients were collected from May 2019 through May 31, 2022. Samples were detected on an 8-color BD FACSCanto II flow cytometer, and analyzed by FACS Diva software from BD or Kaluza software from Beckman Coulter. Antigen full expression, partial expression and no expression were defined as >80%, 20%-80% and <20% of gated cells displaying the antigen of interest, respectively. A total of 274 r/r B-ALL patients planned to be administered CD19 CAR-T cells were assayed CD58 antigen before CAR-T, all of them had received multiline chemotherapies, 61 (22.3%) underwent allogeneic hematopoietic cell transplant (allo-HCT) and 6 cases had a history of antibody (CD20, CD19 or CD22) treatment. The median age was 12 years (range, 1-75 years), including 97 adults and 177 children younger than 18 years. Among all 274 patients, 228 (83.2%, 228/274) showed CD58 expression, consisting of 180 (78.9%, 180/228) cases with full expression and 48 (21.1%, 48/228) with partial expression; whereas 46 (16.8%, 46/274) patients were CD58-negative, demonstrating that CD58 was expressed on the majority (83.3%) of r/r B-ALL patients after treated by multiple intensive chemo drugs and even allo-HCT. Then, we observed the influence of CAR-T cells on the expression of CD58 antigen of lymphoblasts. We followed-up 57 patients who had CD58 expression before CD19 CAR-T but failed or relapsed after CAR-T. Of them, 45.6% (26/57) cases received the further treatment as a consolidation following CD19 CAR-T (CD22 CAR-T, 14; allo-HCT, 12). Among 57 patients, 10 were CD58-negative while 47 cases still showed CD58-positive, the frequency of CD58 expression was 82.5% (47/57) after treated by CAR-T cells and allo-HCT. When we excluded the 26 patients who received following CD22 CAR-T or allo-HCT, and only considered the 31 patients exposed to one-time CD19 CAR-T cells, the CD58 expression rate was 87.1% (27/31). It was reported that CD58 loss in tumor cells induced resistance to CAR-T therapy in cell lines and mouse models. We therefore analyzed the influence of CD58 expression on the treatment response of CAR-T cells. Among 274 r/r B-ALL patients received CD19 CAR-T, 6 were unable to evaluate the treatment response due to death or hospital discharge within 15 days after cell infusion, the remaining 268 patients had evaluation data including 223 cases with CD58 expression (full or partial expression) and 45 without CD58 expression. After CD19 CAR-T therapy, the CR (including MRD+CR) rate in patients with CD58 expression was 91.9% (205/223) while it was 95.6% (43/45) in patients without CD58 expression, indicating that CD58 negativity was irrelevant to CAR-T treatment failure in r/r B-ALL. In conclusion, CD58 antigen was expressed in the majority (83.2%) of r/r B-ALL cells without exposing to CAR-T treatment, and still expressed on the lymphoblasts of most patients (>80%) failed or relapsed after CAR-T, therefore, it could be as a leukemic marker for identifying tumor cells in r/r B-ALL patients who have received multiline therapies including allo-HCT and CAR-T. Additionally, CD58-negative patients achieved a similar high CR rate as CD58-positive patients did after CD19 CAR-T.

Translatome Proteomics Identifies Autophagy As a Resistance Mechanism to on-Target FLT3 Inhibitors in Acute Myeloid Leukemia

Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 are among the most frequent mutational targets in acute myeloid leukemia (AML), drive leukemia progression and confer a poor prognosis. Primary resistance to FLT3 kinase inhibitors (FLT3i) such as quizartinib, crenolanib and gilteritinib is a frequent clinical challenge and often occurs in the absence of identifiable genetic causes, suggesting that adaptive cellular mechanisms mediate primary resistance to on-target FLT3i therapy. We therefore systematically investigated acute cellular responses to on-target therapy with multiple FLT3i in FLT3-ITD+ AML using recently developed translatome proteomics to measure changes in the nascent proteome in combination with kinase signaling mapping by phosphoproteomics to identify therapy resistance mechanisms to FLT3 inhibitors in vitro. This systems-biology approach showed that the autophagy network escapes global translation repression caused by FLT3i and pinpointed autophagy induction mediated by the AKT-mTORC1-ULK1 axis as the most dominant stress response mechanism to on-target FLT3i therapy. FLT3i induced autophagy in a concentration- and time-dependent manner specifically in FLT3-ITD+ cells in vitro and in primary human AML cells ex vivo. Inhibition of FLT3-ITD signaling led to decreased phosphorylation at AKT S473, decreased phosphorylation at the AKT substrate mTOR S2448, and decreased phosphorylation at the mTORC1 substrate ULK1 S757. CRISPR/Cas9-targeting of TSC2, a negative mTORC1 regulator, ULK1 or ATG3, a core autophagy protein required for autophagosome formation, significantly attenuated or largely abolished autophagy induction by FLT3i. Pharmacological autophagy inhibition (ROC-325, Lys05) or genetic ablation of autophagy (shRNA-mediated knockdown of ATG3, ULK1 or its target Beclin1) increased the sensitivity to FLT3-targeted therapy in cell lines, patient-derived xenografts and primary AML cells ex vivo. In immunodeficient mice xenografted with human FLT3-ITD+ AML cells, oral co-treatment with gilteritinib and autophagy inhibitor ROC-325 synergistically impaired leukemia progression and extended overall survival. Our findings show the utility of functional translatome proteomics to systematically investigate cell-autonomous therapy resistance mechanisms, identify a molecular mechanism responsible for primary on-target FLT3i treatment resistance and demonstrate the pre-clinical efficacy of a rational combination treatment strategy targeting both FLT3 and autophagy induction.

Mitochondrial dysfunction and impaired growth of glioblastoma cell lines caused by antimicrobial agents inducing ferroptosis under glucose starvation

Glioblastoma is a difficult-to-cure disease owing to its malignancy. Under normal circumstances, cancer is dependent on the glycolytic system for growth, and mitochondrial oxidative phosphorylation (OXPHOS) is not well utilized. Here, we investigated the efficacy of mitochondria-targeted glioblastoma therapy in cell lines including U87MG, LN229, U373, T98G, and two patient-derived stem-like cells. When glioblastoma cells were exposed to a glucose-starved condition (100 mg/l), they rely on mitochondrial OXPHOS for growth, and mitochondrial translation product production is enhanced. Under these circumstances, drugs that inhibit mitochondrial translation, called antimicrobial agents, can cause mitochondrial dysfunction and thus can serve as a therapeutic option for glioblastoma. Antimicrobial agents activated the nuclear factor erythroid 2-related factor 2–Kelch-like ECH-associated protein 1 pathway, resulting in increased expression of heme oxygenase-1. Accumulation of lipid peroxides resulted from the accumulation of divalent iron, and cell death occurred via ferroptosis. In conclusion, mitochondrial OXPHOS is upregulated in glioblastoma upon glucose starvation. Under this condition, antimicrobial agents cause cell death via ferroptosis. The findings hold promise for the treatment of glioblastoma.

Translatome proteomics identifies autophagy as a resistance mechanism to on-target FLT3 inhibitors in acute myeloid leukemia

Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 occur in 25 % of acute myeloid leukemia (AML) patients, drive leukemia progression and confer a poor prognosis. Primary resistance to FLT3 kinase inhibitors (FLT3i) quizartinib, crenolanib and gilteritinib is a frequent clinical challenge and occurs in the absence of identifiable genetic causes. This suggests that adaptive cellular mechanisms mediate primary resistance to on-target FLT3i therapy. Here, we systematically investigated acute cellular responses to on-target therapy with multiple FLT3i in FLT3-ITD + AML using recently developed functional translatome proteomics (measuring changes in the nascent proteome) with phosphoproteomics. This pinpointed AKT-mTORC1-ULK1-dependent autophagy as a dominant resistance mechanism to on-target FLT3i therapy. FLT3i induced autophagy in a concentration- and time-dependent manner specifically in FLT3-ITD + cells in vitro and in primary human AML cells ex vivo. Pharmacological or genetic inhibition of autophagy increased the sensitivity to FLT3-targeted therapy in cell lines, patient-derived xenografts and primary AML cells ex vivo. In mice xenografted with FLT3-ITD + AML cells, co-treatment with oral FLT3 and autophagy inhibitors synergistically impaired leukemia progression and extended overall survival. Our findings identify a molecular mechanism responsible for primary FLT3i treatment resistance and demonstrate the pre-clinical efficacy of a rational combination treatment strategy targeting both FLT3 and autophagy induction.

Antiviral Evaluation of UV-4B and Interferon-Alpha Combination Regimens against Dengue Virus.

Dengue virus (DENV) is a flavivirus associated with clinical manifestations ranging in severity from self-limiting dengue fever, to the potentially life threatening condition, severe dengue. There are currently no approved antiviral therapies for the treatment of DENV. Here, we evaluated the antiviral potential of four broad-spectrum antivirals, UV-4B, interferon-alpha (IFN), sofosbuvir (SOF), and favipiravir (FAV) against DENV serotype 2 as mono- and combination therapy in cell lines that are physiologically relevant to human infection. Cell lines derived from human liver (HUH-7), neurons (SK-N-MC), and skin (HFF-1) were infected with DENV and treated with UV-4B, IFN, SOF, or FAV. Viral supernatant was sampled daily and infectious viral burden was quantified by plaque assay on Vero cells. Drug effect on cell proliferation in uninfected and infected cells was also assessed. UV-4B inhibited DENV in HUH-7, SK-N-MC, and HFF-1 cells yielding EC50 values of 23.75, 49.44, and 37.38 µM, respectively. Clinically achievable IFN concentrations substantially reduced viral burden in HUH-7 (EC50 = 102.7 IU/mL), SK-N-MC (EC50 = 86.59 IU/mL), and HFF-1 (EC50 = 163.1 IU/mL) cells. SOF potently inhibited DENV in HUH-7 cells but failed to produce the same effect in SK-N-MC and HFF-1 cells. Finally, FAV provided minimal suppression in HUH-7 and SK-N-MC cells, but was ineffective in HFF-1 cells. The two most potent anti-DENV agents, UV-4B and IFN, were also assessed in combination. UV-4B + IFN treatment enhanced antiviral activity in HUH-7, SK-N-MC, and HFF-1 cells relative to monotherapy. Our results demonstrate the antiviral potential of UV-4B and IFN against DENV in multiple physiologically relevant cell types.

Effect of berberine associated with photodynamic therapy in cell lines.

Cervical cancer is a serious worldwide health problem. In view of the potentially harmful effects of current conventional therapies, photodynamic therapy may be an option as it is a minimally invasive therapy and can promote selective cytotoxic activity for neoplastic cells in the target tissue., Berberine (BBR) as an isolated molecule is a natural compound that has antineoplastic properties and potential action as a photosensitizer agent. The purpose of this study was to evaluate the use of berberine as a photosensitizer in photodynamic therapy (PDT) protocols and observe the effects produced by this association in cervical carcinoma cells and in immortalized keratinocytes. Incubation with 2.5 μM berberine promoted less than 10 % of cellular death in both cell lines studied. In addition, by fluorescence microscopy, we demonstrated that berberine was internalized by the cells, and after a period of 48 h, it was still present in the intracellular environment preferentially localized in the cytoplasm. After photodynamic therapy using berberine as a photosensitizer and visible light activation at 447 (±10) nm, we observed a phototoxic effect, which resulted in 19.84 % cell viability for Caski cells and 47.22 % cell viability for HaCaT. Treatment with berberine associated with photodynamic therapy promoted an increase in the production of reactive species of oxygen (ROS) and caspase-3 activity, indicating a preferential cell death mechanism by caspase-dependent apoptosis. Therefore, we demonstrated that berberine is an efficient photosensitizer and that its association with photodynamic therapy may be a potential anticancer treatment strategy for cervical cancer.

Abstract 3587: Targeting cancer metabolism: A novel approach for improved radiotherapy

Abstract One key distinguishing hallmark of cancer cells includes a deregulated cellular metabolism that can be reprogrammed to preferentially exhibit dependence on glycolysis over oxidative phosphorylation (OXPHOS) even in the presence of oxygen (commonly known as “Aerobic Glycolysis” or the Warburg Effect). The bi-directional conversion of glucose to lactate in the presence of oxygen is mainly driven by the activity of the lactate dehydrogenase enzyme (LDH), of which, its A isoform is highly overexpressed in a variety of tumors. In the present study we characterized two novel LDH inhibitors (NCI-006 and NCI-737) in combination with ionizing radiation (IR) for their anti-cancer and radio-sensitization effects across tumor types. This study primarily examined effects of this novel combination therapy in cell lines of Pancreatic (MiaPaca, Hs766T, Panc-1), Lung (H460, A549), Head and Neck (FaDU, UMSCC-1), and Prostate (DU145, PC3) origin. Our preliminary results indicated that targeting LDH in conjunction with IR can enhance radiosensitivity under both hypoxic and normoxic conditions across glycolytic tumor cell lines while not affecting non-glycolytic/normal cells (1522, skin fibroblast) in vitro. Further we established that this enhanced radiosensitivity could be attributed to reduced DNA repair as seen by enhanced expression of y-H2AX and reduced ATP generation. Our results also indicated that increased expression of lactate and glucose transporters, combined with cellular bioenergetics changes, leads to a reprogramming of cells to OXPHOS. Lastly, we found that inhibition of OXPHOS and NAD+ in conjunction with these novel LDH inhibitors promotes metabolic synthetic lethality in vitro. As cancer patients continue to receive radiation for local tumor control, we hope that future studies targeting cancer metabolic vulnerabilities in combination with IR in vivo will enhance the therapeutic ratio of radiotherapy. *The LDH inhibitors used in this study were provided by the NCI Experimental Therapeutics (NExT) Program Citation Format: Suchet Taori, Sarwat Naz, Janet Gamson, Askale Mathias, John Cook, James B. Mitchell. Targeting cancer metabolism: A novel approach for improved radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3587.

Abstract 5167: Stem-like colorectal cancer cell lines show response to the ERK1/2 inhibitor, SCH772984, alone and in combination with neratinib

Abstract Background: We have recently shown an association of colorectal tumor subtypes with differential response to chemotherapy in patients (pts), and to targeted therapy in cell lines. Pts enrolled in NSABP/NRG C-07 with stem-like tumors had a poor prognosis regardless of stage or treatment, highlighting the importance of finding new treatments for stem-like tumors. Our in-vitro data showed that KRAS mutant (mt) cell lines of the intrinsic inflammatory subtype were sensitive to the combination of MEK-162 and neratinib, but stem-like subtype cell lines were resistant regardless of KRAS mt status. The purpose of this study was to extend our observations to xenograft models and to identify new agents that target the stem-like subtype. Methods: KRAS mt cell lines of the inflammatory (NCI-H747) or stem-like (SW-480) subtype were used in xenograft models to test inhibition of tumor growth by MEK-162 and neratinib, alone or in combination. Five stem-like cell lines with KRAS wt (C2BBE1, HS675T) or KRAS mt (SW480, SW620, HCT116), and two KRAS mt inflammatory cell lines (NCI-H747, SW837) were tested for cell viability after treatment with SCH772984 alone or in combination with neratinib. Results: In vivo analysis showed that treatment of NCI-H747 xenografts with a combination of MEK-162 (3mg/kg) and neratinib (10mg/kg) led to significant tumor regression compared to treatment with either drug alone (p ≥0.0001 v neratinib alone; p ≥0.002 v MEK-162 alone). In contrast, MEK-162 alone was able to inhibit tumor growth of stem-like cell line (SW480) xenografts (p ≥0.0061 v control) but neratinib was ineffective (p ≥0.145 v control) as a single agent and did not add to MEK-162. Our in-vitro and in-vivo data showed that inhibition of p-ERK directly correlated with sensitivity to the MEK and neratinib combination in inflammatory subtype. SCH772984 significantly inhibited cell viability of both inflammatory (IC50 1-2 μM) and stem-like subtype (IC50 1-2 μM) cell lines. The combination of neratinib (0.125 μM) and SCH772984 (1 μM) was effective at decreasing cell viability by 60-70% in both inflammatory and stem-like cell lines. Inhibition of ERK phosphorylation was correlated with loss of cell viability. Conclusion: The combination of MEK-162 and neratinib work synergistically to decrease tumor growth in inflammatory but not stem-like colorectal cancer subtypes. We demonstrate that SCH772984 decreases the viability of stem-like colon cancer cell lines, and works synergistically with neratinib. The use of SCH772984 alone, but more potently in combination with neratinib, may represent a therapeutic approach for pts with stem-like tumors, a finding that underscores the potential importance of employing subtype analysis in the diagnosis of colon cancer and potentially as to a guide to new therapies. Support: PA DoH, which disclaims certain responsibility. Citation Format: Rekha Pal, Ning Wei, Nan Song, Shao-yu Wu, S. Rim Kim, Patrick G. Gavin, Peter C. Lucas, Ashok Srinivasan, Samuel A. Jacobs, Soonmyung Paik, John C. Schmitz, Kay Pogue-Geile. Stem-like colorectal cancer cell lines show response to the ERK1/2 inhibitor, SCH772984, alone and in combination with neratinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5167. doi:10.1158/1538-7445.AM2017-5167

High expression of cholesterol biosynthesis genes is associated with resistance to statin treatment and inferior survival in breast cancer.

There is sufficient evidence that statins have a protective role against breast cancer proliferation and recurrence, but treatment predictive biomarkers are lacking. Breast cancer cell lines displaying diverse sensitivity to atorvastatin were subjected to global transcriptional profiling and genes significantly altered by statin treatment were identified. Atorvastatin treatment strongly inhibited proliferation in estrogen receptor (ER) negative cell lines and a commensurate response was also evident on the genome-wide transcriptional scale, with ER negative cells displaying a robust deregulation of genes involved in the regulation of cell cycle progression and apoptosis. Interestingly, atorvastatin upregulated genes involved in the cholesterol biosynthesis pathway in all cell lines, irrespective of sensitivity to statin treatment. However, the level of pathway induction; measured as the fold change in transcript levels, was inversely correlated to the effect of statin treatment on cell growth. High expression of cholesterol biosynthesis genes before treatment was associated with resistance to statin therapy in cell lines and clinical biopsies. Furthermore, high expression of cholesterol biosynthesis genes was independently prognostic for a shorter recurrence-free and overall survival, especially among ER positive tumors. Dysregulation of cholesterol biosynthesis is therefore predictive for both sensitivity to anti-cancer statin therapy and prognosis following primary breast cancer diagnosis.

Abstract 1836: Erlotinib induces NF-kappa B dependence that promotes EGFR tyrosine kinase inhibitor resistance in lung adenocarcinoma

Abstract The vast majority of patients with lung adenocarcinomas harboring activating mutation in EGFR respond to EGFR tyrosine inhibitors (TKI) (i.e. erlotinib). However, the magnitude of tumor regression is variable and responses are short-lived with a median duration of 9-12 months. We recently identified activation of the NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) signaling pathway as a key mediator of de novo resistance to EGFR TKI therapy in cell lines and tumor xenograft models of lung adenocarcinoma. However, the role of NF-κB activation in mediating EGFR TKI acquired resistance in lung adenocarcinoma is not well defined. We have found that erlotinib treatment of EGFR TKI sensitive lung adenocarcinoma cell lines promotes the rapid phosphorylation and degradation of IκB (inhibitor of NF-κB) and subsequent activation of the NF-κB subunit RelA. RelA activation is accompanied by transcriptional activation of downstream NF-κB target genes, including IL6. Prolonged exposure of EGFR TKI sensitive lung adenocarcinoma cell lines to erlotinib leads to EGFR TKI acquired resistance that is accompanied by sustained NF-κB activation and IL6 expression. EGFR TKI acquired resistance can be overcome by treating cells with erlotinib in combination with PBS-1086 (rel∼MD, Inc.), a direct Rel inhibitor. Furthermore, concomitant treatment of EGFR TKI sensitive lung adenocarcinoma cells with erlotinib + PBS-1086 prevents the development of EGFR TKI acquired resistance. Together, these results demonstrate the molecular basis for the synthetic lethality of combined EGFR and NF-κB inhibition and provide mechanism-based rationale for polytherapies against both EGFR and NF-κB to enhance response in lung adenocarcinoma patients. Broadly, our findings provide novel insights into the biological and clinical consequences of the context-specific and dynamic functional interplay between EGFR and NF-κB signaling. Citation Format: Collin M. Blakely, Evangelos Pazarentzos, Saurabh Asthana, Victor Olivas, Irena Tan, Timothy Fouts, Jeffrey Meshulam, Trever G. Bivona. Erlotinib induces NF-kappa B dependence that promotes EGFR tyrosine kinase inhibitor resistance in lung adenocarcinoma. [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 1836. doi:10.1158/1538-7445.AM2014-1836

Abstract 4631: Pharmacologic inhibition of NF-kappaB overcomes de novo resistance to erlotinib in models of EGFR-mutant lung adenocarcinoma.

Abstract Treatment of patients with lung cancers that harbor activating mutations in the kinase domain of EGFR (e.g. L858R, in frame Exon19 deletion), which account for 15-20% of lung adenocarcinomas, with the EGFR tyrosine kinase inhibitor (TKI) erlotinib, causes tumor regression in many patients. However, the magnitude of tumor regression is variable and responses are short-lived with a median duration of 9-12 months. Thus inhibition of mutant EGFR activity by treatment with erlotinib results in incomplete responses and is not curative in the majority of EGFR-mutant lung cancer patients. While mechanisms of acquired resistance to EGFR TKI treatment are well defined, mechanisms of de novo resistance, or incomplete initial response to EGFR TKI therapy, remain uncharacterized. We recently identified activation of the NF-kB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) signaling pathway as a key mediator of de novo resistance to EGFR TKI therapy in cell lines and tumor xenograft models of lung cancer. Furthermore, patients with EGFR-mutant lung cancers that demonstrate elevated IkB (NF-kB inhibitor) expression, and consequently low NF-kB activity, exhibit improved responses to EGFR TKIs compared to patients with low IkB levels. NF-kB is a family of transcription factors that regulates numerous biological processes, including cell proliferation and apoptosis, as well as a critical mediator of both innate and adaptive immune cell activity. We hypothesized that pharmacologic inhibition of NF-kB activity would enhance the efficacy of EGFR TKI in EGFR-mutant lung cancer. Using a novel rel (reticuloendotheliosis) inhibitor of NF-kB, PBS-1086 (rel∼MD, Inc.), we found that treatment of human lung cancer cells and tumor xenografts that harbor an EGFRL858R activating mutation (11-18), yet are EGFR TKI resistant, with erlotinib in combination with PBS-1086 induces tumor cell apoptosis and tumor regression. Erlotinib + PBS-1086 treatment results in decreased binding of the NF-kB subunit RelA to the IL6 promoter and a significant decrease in IL6 mRNA expression. IL6 has previously been shown to promote EGFR-mutant lung adenocarcinoma cell proliferation and survival, and its downregulation by PBS-1086 represents a possible mechanism of action of the compound. Using CC10-rtTA; Tet-Op-EGFR-mutant transgenic mouse models of lung adenocarcinoma, we have shown that treatment of mice with PBS-1086 enhances erlotinib-induced tumor regression. We believe that these results will guide future clinical trials of NF-kB inhibitors in combination with erlotinib to improve outcomes in selected EGFR-mutant lung cancer patients. Citation Format: Collin M. Blakely, Victor Olivas, Jie Zhang, Trever G. Bivona. Pharmacologic inhibition of NF-kappaB overcomes de novo resistance to erlotinib in models of EGFR-mutant lung adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4631. doi:10.1158/1538-7445.AM2013-4631

Improving Endocrine Therapy for Breast Cancer: It's Not That Simple

Endocrine therapy is the mainstay of treatment for estrogen receptor (ER)-positive breast cancer and often represents the first several lines of treatment in the metastatic setting. Unfortunately both de novo and acquired resistance to endocrine therapy remain important clinical problems, and recent efforts have centered around strategies to combat this resistance. Preclinical studies implicate crosstalk between ER and critical signaling pathways, such as the epidermal growth factor receptor/human epidermal growth factor receptor 2 (HER2) and extracellular signal-regulating kinase 1/2/mitogen activated protein kinase cascade and the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway, as key mediators of endocrine resistance. Focus on the PI3K/AKT/mTOR pathway has intensified recently, not only because PI3K-activating mutations are found in 36% of human breast cancers, but also based on preclinical studies associating activation of the PI3K/AKT/mTOR pathway with endocrine resistance, and correlating mTOR inhibition with sensitivity to endocrine therapy in cell lines. In addition, estrogen deprivation increases apoptosis following PI3K inhibitor treatment, providing a rationale for combined therapy even in endocrine-sensitive disease. For all of these reasons inhibition of the PI3K/mTOR pathway, both alone and in combination with antiestrogen therapies, is an active area of research in the treatment of ER-positive disease, which still kills more women than any other breast cancer subset. Clinical testament to the value of cotargeting ER and the PI3K/ AKT/mTOR pathway comes from several key papers. In the Breast Cancer Trials of Oral Everolimus-2 (BOLERO-2), a randomized phase III study of 724 postmenopausal women with hormone receptor (HR) –positive, HER2-negative metastatic breast cancer that had progressed on therapy with a nonsteroidal aromatase inhibitor (AI), combination therapy with exemestane and the mTOR inhibitor everolimus resulted in significantly longer progression-free survival (PFS; 7.8 months v 3.2 months) and higher response rate than singleagent exemestane. In the Tamoxifen and RAD001 (TAMRAD) study, a smaller randomized phase II trial in a similar patient population, patients receiving the combination of tamoxifen and everolimus had a higher clinical benefit rate (61%) and longer time to progression (8.6 months) than the group receiving tamoxifen alone (42% and 4.5 months). In that study, patients with secondary resistance to AI seemed to benefit more from the combination than patients with primary resistance, supporting the concept that cross-talk between pathways and the sequential upregulation of the PI3K/mTOR signaling is a mechanism of resistance. While these two studies each used a different endocrine agent, they were otherwise similar in several key respects: the population studied had evidence of acquired AI resistance, both used the mTOR inhibitor everolimus, and improvements in clinical end points were similar, prompting the US Food and Drug Administration to approve everolimus for second-line treatment of HR-positive breast cancer in July 2012. However these benefits were not without toxicity; in both studies combined everolimus/endocrine therapy had substantially higher stomatitis (56%), rash (36% to 44%), fatigue (33% to 72%), diarrhea (30% to 39%), and anorexia (29% to 43%) than single-agent endocrine therapy. Given real toxicity and substantial cost ( $5,000 per month), clinical decision making regarding the addition of everolimus to endocrine therapy is generally made on a case-by-case basis. In the article accompanying this editorial, Wolff et al examine the combination of the nonsteroidal AI, letrozole, and the mTOR inhibitor, temsirolimus, in the first-line treatment of metastatic breast cancer. This study, called HORIZON, was a large multicenter multinational randomized phase III trial involving 1,112 patients, that found no benefit in PFS associated with adding the mTOR inhibitor to AI therapy. Indeed, the trial was stopped for futility by the independent data monitoring committee. Response rate and overall survival were also similar between groups. Interestingly, although the combination arm had higher incidence of expected mTOR inhibitor–related toxicity than the single-agent arm, the occurrence of these toxicities was lower than that seen in the everolimus studies described above. For example only 14% of patients experienced mucositis, 15% rash, and 21% diarrhea. In a subgroup analysis, the authors found that patients 65 years old and younger appeared to benefit from the combination therapy whereas older patients did not. This exploratory but provocative finding was not seen in the subgroup analyses by age in BOLERO-2, and the median age was similar in both studies. Why would these large trials of combination therapy with mTOR inhibitors and AIs yield discordant results? One key difference is that the patient population in the HORIZON study was largely AI-naive and temsirolimus/letrozole was the first endocrine treatment for metastatic disease. By contrast, patients in both BOLERO-2 and TAMRAD had AI-resistant disease. In BOLERO-2, 84% had initially endocrinesensitive disease and then progressed on an AI, so this population was essentially the same secondary resistance population that showed the JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 31 NUMBER 2 JANUARY 1

Novel 2'-substituted, 3'-deoxy-phosphatidyl-myo-inositol analogues reduce drug resistance in human leukaemia cell lines with an activated phosphoinositide 3-kinase/Akt pathway.

Activation of the phosphoinositide 3-kinase (PI3-K)/Akt signalling pathway has been linked with resistance to chemotherapeutic drugs, and its down-regulation, by means of pharmacological inhibitors of PI3-K, considerably lowers resistance to various types of therapy in cell lines derived from solid tumours. Recently, a new class of Akt inhibitors, referred to as phosphatidylinositol ether lipids (PIAs), have been synthesized. We tested whether two new PIAs could lower the sensitivity threshold to chemotherapeutic drugs of human leukaemia cell lines with an activated PI3-K/Akt network. We used HL60AR (for apoptosis resistant), K562 and U937 cells. The two pharmacological inhibitors, used at 5 micromol/l, down-regulated Akt kinase activity and phosphorylation. Neither of the two chemicals affected the activity of other signalling proteins in the Akt pathway, such as phosphoinositide-dependent protein kinase-1 or PTEN. When employed at 5 micromol/l, the Akt inhibitors markedly reduced the resistance of the leukaemic cell lines to etoposide or cytarabine. Remarkably, a 5 micromol/l concentration of the inhibitors did not negatively affect the survival rate of human cord blood CD34(+) cells. Overall, our results indicate that new selective Akt pharmacological inhibitors might be used in the future for overcoming Akt-mediated resistance to therapeutic treatments of acute leukaemia cells.