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Supplementary Data from In Vivo Syngeneic Tumor Models with Acquired Resistance to Anti–PD-1/PD-L1 Therapies

Antibodies targeting PD-1 and PD-L1 have produced durable responses in a subset of patients with cancer. However, a majority of these patients will ultimately relapse due to acquired resistance. To explore the underlying mechanisms of this secondary resistance, we developed five syngeneic murine tumor variants with acquired resistance to anti–PD-1 and/or PD-L1 antibodies in vivo. Resistant in vivo models were obtained by serial treatment/reimplantation cycles of the MC38 colorectal, MB49 and MBT2 bladder, and RENCA kidney and TyrNras melanoma models. Tumor immune infiltrates were characterized for wild type and resistant tumors using spectral cytometry and their molecular alterations analyzed using RNA sequencing analyses. Alterations in the tumor immune microenvironment were strongly heterogeneous among resistant models, involving select lymphoid and/or myeloid subpopulations. Molecular alterations in resistant models included previously identified pathways as well as novel candidate genes found to be deregulated in several resistant models. Among these, Serpinf1, coding for pigment epithelial-derived factor (PEDF) was further explored in the MC38 and the MBT2 models. Overexpression of Serpinf1 induced resistance to anti–PD-1 antibodies in the MC38 model, whereas knockdown of Serpinf1 sensitized this model as well as the primarily resistant MBT2 model. Serpinf1 overexpression was associated with increased production of free fatty acids and reduced activation of CD8+ cells, while orlistat, a compound that reduces the production of free fatty acids, reversed resistance to anti–PD-1 therapy. Our results suggest that a panel of syngeneic resistant models constitutes a useful tool to model the heterogeneity of resistance mechanisms encountered in the clinic.

Supplementary figure legends for supplementary figures 1,2 and 3

Direct cell death induction, in addition to immune-effector cell-mediated mechanisms, is one of the key mechanisms of action of anti-CD20 antibodies, and yet the signaling pathways implicated remain poorly investigated. Here we show that the transcription factor EGR-1 is rapidly induced by anti-CD20 antibodies and is a key mediator for CD20-induced cell death. EGR-1 induction results from an increased calcium influx induced by anti-CD20 antibodies. We show that both rituximab and obinutuzumab induce calcium influx, albeit through different mechanisms, and this influx is crucial for cell death induction. Inhibition of the calcium flux with calcium channel blockers (CCB) abolished EGR-1 induction and impaired the efficacy of anti-CD20 antibodies in preclinical in vitro and in vivo models. Finally, we investigated the impact of CCBs in patients treated with anti-CD20 antibodies included in the clinical trials GOYA and REMARC, and found that patients simultaneously receiving CCBs and anti-CD20 therapy have a shorter progression-free survival and overall survival. These results reveal EGR-1 as a key mediator of the direct cytotoxic activity of anti-CD20 antibodies and provide a rationale to evaluate EGR-1 expression as a new biomarker to predict response to anti-CD20 treatment. In addition, our findings show that calcium influx is required for anti-CD20–mediated tumor cell death and suggest that simultaneous administration of calcium channel blocking agents could be deleterious in patients receiving anti-CD20–based immunotherapy.

The tumor growth inhibition (TGI) was calculated on day 59 and 69 for untreated group and treated groups, respectively. The control group was sacrificed at day 59 as the tumor reached the maximum tolerated tumor size (2000 mm3) and the treated groups were maintained until day 69. In this preliminary experiment we demonstrated a potentiation of rituximab antitumor activity and extended mice survival when co-injected with pegfilgrastim. The results are reported and displayed as the mean {plus minus} SEM of six mice per group

The tumor growth inhibition (TGI) measured on day 56 confirmed the potentiation of trastuzumab antitumor activity when co-injected with pegfilgrastim. Co-treatment with pegfilgrastim and trastuzumab significantly decreased A549 growth when compared to untreated, trastuzumab alone and filgrastim plus trastuzumab groups according to one way ANOVA (p

Supplementary Figures

PDF file - 106K, Supplemental table 1. Antibodies used in the study; Supplemental table 2. Genes differentially expressed in MM1.S cells exposed to IGF-1 (200 ng/mL) and bortezomib (10 nM). The list indicate genes with at least a 2-fold difference in expression levels (Agilent pangenomic array)

Purpose: Multiple myeloma is a clonal plasma cell disorder in which growth and proliferation are linked to a variety of growth factors, including insulin-like growth factor type I (IGF-I). Bortezomib, the first-in-class proteasome inhibitor, has displayed significant antitumor activity in multiple myeloma.Experimental Design: We analyzed the impact of IGF-I combined with proteasome inhibitors on multiple myeloma cell lines in vivo and in vitro as well as on fresh human myeloma cells.Results: Our study shows that IGF-I enhances the cytotoxic effect of proteasome inhibitors against myeloma cells. The effect of bortezomib on the content of proapoptotic proteins such as Bax, Bad, Bak, and BimS and antiapoptotic proteins such as Bcl-2, Bcl-XL, XIAP, Bfl-1, and survivin was enhanced by IGF-I. The addition of IGF-I to bortezomib had a minor effect on NF-κB signaling in MM.1S cells while strongly enhancing reticulum stress. This resulted in an unfolded protein response (UPR), which was required for the potentiating effect of IGF-I on bortezomib cytotoxicity as shown by siRNA-mediated inhibition of GADD153 expression.Conclusions: These results suggest that the high baseline level of protein synthesis in myeloma can be exploited therapeutically by combining proteasome inhibitors with IGF-I, which possesses a “priming” effect on myeloma cells for this family of compounds. Clin Cancer Res; 19(13); 3556–66. ©2013 AACR.

Supplementary Figure 1 from Silencing of Tubulin Binding Cofactor C Modifies Microtubule Dynamics and Cell Cycle Distribution and Enhances Sensitivity to Gemcitabine in Breast Cancer Cells

PDF file - 464K, Results shown are representative of three separate experiments (S1C, S2, S3A) and mean values from 3 independent experiments +/- SD (S1A-B, S4)

Supplementary Figure 3 from Silencing of Tubulin Binding Cofactor C Modifies Microtubule Dynamics and Cell Cycle Distribution and Enhances Sensitivity to Gemcitabine in Breast Cancer Cells

Supplementary Figure Legend for Supplementary Figure 1 from Silencing of Tubulin Binding Cofactor C Modifies Microtubule Dynamics and Cell Cycle Distribution and Enhances Sensitivity to Gemcitabine in Breast Cancer Cells

PDF file - 69K

Supplementary Figure 2 from Silencing of Tubulin Binding Cofactor C Modifies Microtubule Dynamics and Cell Cycle Distribution and Enhances Sensitivity to Gemcitabine in Breast Cancer Cells

Tubulin binding cofactor C (TBCC) is essential for the proper folding of α- and β-tubulins into microtubule polymerizable heterodimers. Because microtubules are considered major targets in the treatment of breast cancer, we investigated the influence of TBCC silencing on tubulin pools, microtubule dynamics, and cell cycle distribution of breast cancer cells by developing a variant MCF7 cells with reduced content of TBCC (MC−). MC− cells displayed decreased content in nonpolymerizable tubulins and increased content of polymerizable/microtubule tubulins when compared with control MP6 cells. Microtubules in MC− cells showed stronger dynamics than those of MP6 cells. MC− cells proliferated faster than MP6 cells and showed an altered cell cycle distribution, with a higher percentage in S-phase of the cell cycle. Consequently, MC− cells presented higher sensitivity to the S-phase–targeting agent gemcitabine than MP6 cells in vitro. Although the complete duration of mitosis was shorter in MC− cells and their microtubule dynamics was enhanced, the percentage of cells in G2-M phase was not altered nor was there any difference in sensitivity to antimicrotubule-targeting agents when compared with MP6 cells. Xenografts derived from TBCC variants displayed significantly enhanced tumor growth in vivo and increased sensitivity to gemcitabine in comparison to controls. These results are the first to suggest that proteins involved in the proper folding of cytoskeletal components may have an important influence on the cell cycle distribution, proliferation, and chemosensitivity of tumor cells. Mol Cancer Ther; 10(2); 303–12. ©2011 AACR.

Human erythrocytes represent candidates of choice as carriers for a wide range of drugs due to their unique biophysical and physiological properties. In this study, we used a sonoporation device generating and monitoring acoustic stable cavitation without any addition of contrast or nucleation agents. The device was evaluated for bovine serum albumin (BSA) delivery into human erythrocytes. After determining the adequate hematocrit percentage compatible with the generation of stable cavitation, we determined the optimal sonoporation conditions allowing BSA delivery while preserving erythrocyte integrity. Our results demonstrate that stable cavitation allows efficient delivery of proteins into human erythrocytes with limited lysis of these cells. In conclusion, our study allowed for the development of a stable and regulated cavitation program and the establishment of sonoporation conditions suitable for intracellular protein delivery while maintaining erythrocyte integrity. Additional investigations are needed to move from the proof of concept to a larger-scale application.

Background The existence of a cross-talk between peritumoral adipocytes and cancer cells has been increasingly investigated. Several studies have shown that these adipocytes protect tumor cells from the effect of anticancer agents. Methods To investigate a potential protective effect of adipocyte-conditioned medium on HER2 positive breast cancer cells exposed to tyrosine kinase inhibitors (TKI) such as lapatinib, we analyzed the sensitivity of HER2 positive breast cancer models in vitro and in vivo on SCID mice in the presence or absence of adipocytes or adipocyte-conditioned medium. Results Conditioned medium from differentiated adipocytes reduced the in vitro sensitivity of the HER2+ cell lines BT474 and SKBR3 to TKI. Particularly, conditioned medium abrogated P27 induction in tumor cells by lapatinib but this was observed only when conditioned medium was present during exposure to lapatinib. In addition, resistance was induced with adipocytes derived from murine NIH3T3 or human hMAD cells but not with fibroblasts or preadipocytes. In vivo studies demonstrated that the contact of the tumors with adipose tissue reduced sensitivity to lapatinib. Soluble factors involved in this resistance were found to be thermolabile. Pharmacological modulation of lipolysis in adipocytes during preparation of conditioned media showed that various lipolysis inhibitors abolished the protective effect of conditioned media on tumor cells, suggesting a role for adipocyte lipolysis in the induction of resistance of tumor cells to TKI. Conclusions Overall, our results suggest that contact of tumor cells with proximal adipose tissue induces resistance to anti HER2 small molecule inhibitors through the production of soluble thermolabile factors, and that this effect can be abrogated using lipolysis inhibitors.

The underlying mechanisms of acquired resistance to the immune checkpoint inhibitory (ICI) PD-1 and PD-L1 antibodies remain largely unknown and need to be further explored. In this study, we established and analyzed robust ICI resistance in in vivo models in order to identify new mechanisms and genes involved in acquired resistance. Resistant in vivo models were obtained by serial treatment/reimplantation cycles in immunocompetent mice bearing MC38, MB49, MBT2, TyrNRas or RENCA tumors. Spectral cytometry was applied to characterize modifications in the tumor immune microenvironment. RNAseq analyses were performed to identify differences in gene expression profiles between sensitive parental models compared to their resistant counterparts. Alterations in the tumor immune microenvironment were highly heterogeneous amongst resistant models, and are thus considered representative for the diversity expected in patients. Each resistant model displayed multiple modifications in the tumor immune infiltrate in comparison to the respective wild type model, involving selected lymphoid and/or myeloid subpopulations. For instance, in anti-PD-1 and anti-PD-L1 resistant MC38 models, we observed a remarkable increase of FoxP3+CD4+ T cells while CD8+ Tem cells were decreased in anti PD-1 resistant models. We also found that the type 2 macrophage content was increased in the anti PD-1 resistant MB49 model. Gene profiling analyses demonstrated the variability associated with acquisition of a resistance phenotype. Alterations of pathways previously reported to be associated with resistance to ICIs were observed in some of our models while other genes appeared to be highly de-regulated in several models. For instance, in the anti PD-L1 resistant MBT2 model many signaling pathways were found to be up-regulated, such as HIF1α, TGFβ, and ERK/MAPK signaling. Several therapeutic combinations, in particular targeting alternative immune checkpoints, were found to reverse the resistance phenotype in selected models. Moreover, in accordance with flow cytometry results, in the anti PD-1 resistant MC38 model we observed a significant delay in tumor growth when a TNFR1 antibody was combined with a PD-1 antibody (p Citation Format: Morgane Denis, Chloé Grasselly, Pierre-Antoine Choffour, Anne Wierinckx, Doriane Mathé, Kamel Chettab, Anne Tourette, Nolan Talhi, Fabian Birzele, Elsa Kress, Lars Petter Jordheim, Christian Klein, Eva-Laure Matera, Charles Dumontet. Characterization of acquired resistant models to therapies targeting the PD-1/PD-L1 axis demonstrates model-dependent mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4200.

International audience; We herein report the development and evaluation of a novel HER2-targeting antibody–drug conjugate (ADC) based on the topoisomerase I inhibitor payload exatecan, using our hydrophilic monodisperse polysarcosine (PSAR) drug-linker platform (PSARlink). In vitro and in vivo experiments were conducted in breast and gastric cancer models to characterize this original ADC and gain insight about the drug-linker structure–activity relationship. The inclusion of the PSAR hydrophobicity masking entity efficiently reduced the overall hydrophobicity of the conjugate and yielded an ADC sharing the same pharmacokinetic profile as the unconjugated antibody despite the high drug-load of the camptothecin-derived payload (drug–antibody ratio of 8). Tra-Exa-PSAR10 demonstrated strong anti-tumor activity at 1 mg/kg in an NCI-N87 xenograft model, outperforming the FDA-approved ADC DS-8201a (Enhertu), while being well tolerated in mice at a dose of 100 mg/kg. In vitro experiments showed that this exatecan-based ADC demonstrated higher bystander killing effect than DS-8201a and overcame resistance to T-DM1 (Kadcyla) in preclinical HER2+ breast and esophageal models, suggesting potential activity in heterogeneous and resistant tumors. In summary, the polysarcosine-based hydrophobicity masking approach allowsfor the generation of highly conjugated exatecan-based ADCs having excellent physicochemical properties, an improved pharmacokinetic profile, and potent in vivo anti-tumor activity.

The development of targeted therapies has drastically improved the outcome of patients with different types of cancer. T‐DM1 (trastuzumab‐emtansine) is an antibody‐drug conjugate used for the treatment of HER2‐positive breast cancer combining the FDA approved mAb (monoclonal antibody) trastuzumab and the microtubule cytotoxic agent DM1 (emtansine). Despite clinical successes achieved by targeted therapies, a large number of patients develop resistance during treatment. To explore mechanisms of resistance to T‐DM1, the MDA‐MB‐361 HER2‐positive breast cancer cell line was exposed in vitro to T‐DM1 in the absence or presence of ciclosporin A. Previously reported mechanisms of resistance such as trastuzumab‐binding alterations, MDR1 upregulation, and SLC46A3 downregulation were not observed in these models. Despite a decrease in HER2 expression at the cell surface, both resistant cell lines remained sensitive to HER2 targeted therapies such as mAbs and tyrosine kinase inhibitors. In addition, sensitivity to DNA damaging agents and topoisomerase inhibitors were unchanged. Conversely resistance to anti‐tubulin agents increased. Resistant cells displayed a decreased content of polymerized tubulin and a decreased content of βIII tubulin although the downregulation of βIII tubulin by siRNA in the parental cell line did not modified the sensitivity to T‐DM1. Both cell lines resistant to T‐DM1 also presented giant aneuploid cells. Several SLC (solute carrier) transporters were found to be differentially expressed in the resistant cells in comparison to parental cells. These results suggest that some characteristics such as increased baseline aneuploidy and altered intracellular drug trafficking might be involved in resistance to T‐DM1.

Despite abundant epidemiological data linking metals to leukemia and other cancers, baseline values of toxic and essential metals in patients with leukemia and the clinical impact of these metals remain unknown. Thus, we sought to quantify metal values in untreated patients with acute myeloid leukemia (AML) and controls and determine the impact of metal values on AML patients' survival. Serum samples from patients with untreated AML and controls at Hospices Civils de Lyon were analyzed and compared for trace metals and copper isotopic abundance ratios with inductively coupled plasma mass spectrometry. Survival analysis was performed as a function of metal values, and a multi-metal score was developed for patients with AML. Serum samples were collected from 67 patients with untreated AML and 94 controls. Most patients had intermediate-risk cytogenetics (63.1%) without FLT3 internal tandem duplication mutations (75.6%) or NPM1 mutations (68.1%). Most metal values differed significantly between AML and control groups. Patients with lower magnesium and higher cadmium values had the worst survival rates, with only 36% surviving at 6 months (P = .001). The adverse prognostic effect of this combination was maintained on multivariate analysis. Based on this, we developed a novel metal score, which accounts for multiple relative abnormalities in the values of five toxic and five essential metals. Patients with a higher metal score had significantly worse survival, which was maintained on multivariate analysis (P = .03). This baseline metal scoring system was also prognostic when we applied it to a separate population of front-line AML patients.

The nucleotide excision repair protein excision repair cross-complementation group 1 (ERCC1) has been repeatedly shown to be involved in the sensitivity of cancer cells to platinum derivatives. In order to better understand this process, we transfected HCT-116 cells with a plasmid encoding ERCC1 and studied their in vitro and in vivo behaviour. No main differences were observed for sensitivity to platinum drugs, DNA repair capacity and clonogenicity in vitro. However, ­ERCC1-transfected HCT-116 cells showed paradoxical behaviour in vivo with increased growth in mice treated with oxaliplatin as compared to untreated mice. The Trop2 protein was identified as being potentially involved in the underlying mechanism for these observations, as it was overexpressed in transfected cells. Our results suggest complex regulation of signalling in cancer cells exposed to cancer drugs.

// Taghreed Hirz 1 , Eva-Laure Matera 1 , Kamel Chettab 1 , Lars Petter Jordheim 1 , Doriane Mathe 1 , Anne Evesque 1 , Justine Esmenjaud 1 , Gilles Salles 2, 3 and Charles Dumontet 1, 4, 5 1 Anticancer Antibody Team, INSERM U1052, CNRS UMR 5286, Cancer Research Center of Lyon, University of Lyon, Lyon, France 2 Hospices Civils de Lyon, Department of Hematology, Pierre-Benite, France 3 Universite Claude Bernard Lyon-1, Lyon, France 4 ProfileXpert, Lyon, France 5 Laboratory of Hematology, Hospices Civils de Lyon, Pierre-Benite, France Correspondence to: Charles Dumontet, email: charles.dumontet@chu-lyon.fr Keywords: neutrophils, lymphoma, chemotherapy, targeted therapy, cell-cell interactions Received: April 11, 2017 Accepted: July 14, 2017 Published: August 18, 2017 ABSTRACT Innate immune cells constitute a substantial proportion of the cells within the tumor microenvironment. Besides the contribution of the microenvironment to tumor proliferation and survival, there is direct evidence that interactions between tumor cells and their microenvironment alter sensitivity to anti-cancer agents. Neutrophils, a key player in the innate immune system, have been less studied than many other immune cells regarding their impact on cancer cell response to anti-cancer agents. In our 2D and 3D coculture systems, human neutrophils and differentiated HL60 cells attenuated the sensitivity of various lymphoma cell lines to several anti-cancer agents, including targeted therapies. Neutrophil-induced protection was dependent on cell-cell interaction between CD11b and ICAM-1 expressed by neutrophils and B cells, respectively and was shown to be Mcl-1-dependent. The protective effect of neutrophils was validated in vivo using immune-compromised mice inoculated with human NHL with our without neutrophils then followed by treatment with chemotherapy. Similar findings were made on primary cells purified from patients with chronic lymphocytic leukemia, treated with fludarabine or targeted agents in the presence of autologous neutrophils. In a clinical study, patients with non-Hodgkin’s lymphoma with increased neutrophil counts displayed a reduced response rate to therapy. These findings reveal a novel protective mechanism of neoplastic B cells involving innate immune cells which could be pharmacologically targeted to enhance the antitumor effect of therapy.

Direct cell death induction, in addition to immune-effector cell-mediated mechanisms, is one of the key mechanisms of action of anti-CD20 antibodies, and yet the signaling pathways implicated remain poorly investigated. Here we show that the transcription factor EGR-1 is rapidly induced by anti-CD20 antibodies and is a key mediator for CD20-induced cell death. EGR-1 induction results from an increased calcium influx induced by anti-CD20 antibodies. We show that both rituximab and obinutuzumab induce calcium influx, albeit through different mechanisms, and this influx is crucial for cell death induction. Inhibition of the calcium flux with calcium channel blockers (CCB) abolished EGR-1 induction and impaired the efficacy of anti-CD20 antibodies in preclinical in vitro and in vivo models. Finally, we investigated the impact of CCBs in patients treated with anti-CD20 antibodies included in the clinical trials GOYA and REMARC, and found that patients simultaneously receiving CCBs and anti-CD20 therapy have a shorter progression-free survival and overall survival. These results reveal EGR-1 as a key mediator of the direct cytotoxic activity of anti-CD20 antibodies and provide a rationale to evaluate EGR-1 expression as a new biomarker to predict response to anti-CD20 treatment. In addition, our findings show that calcium influx is required for anti-CD20–mediated tumor cell death and suggest that simultaneous administration of calcium channel blocking agents could be deleterious in patients receiving anti-CD20–based immunotherapy.

International audience

International audience; Synthetic progesterone and 5α/β-pregnane-3,20-dione derivatives were evaluated as in vitro and in vivo modulators of multidrug-resistance (MDR) using two P-gp-expressing human cell lines, the non-steroidogenic K562/R7 erythroleukaemia cells and the steroidogenic NCI-H295R adrenocortical carcinoma cells, both resistant to doxorubicin. The maximal effect in both cell lines was observed for 7α-O-benzoyloxy,11α(R)-O-tetrahydropyranyloxy-5β-pregnane-3,20-dione 4. This modulator co-injected with doxorubicin significantly decreased the tumour size and increased the survival time of immunodeficient mice xenografted with NCI-H295R or K562/R7 cells.

International audience; Phosphatidylinositide 3-kinases (PI3Ks) are widely expressed enzymes involved in membrane signalization pathways. Attempts to administer inhibitors with broad activity against different isoforms have failed due to toxicity. Conversely the PI3K delta isoform is much more selectively expressed, enabling therapeutic targeting of this isoform. Of particular interest PI3K delta is expressed in human basophils and its inhibition has been shown to reduce anti-IgE induced basophil degranulation, suggesting that PI3K delta inhibitors could be useful as anti-allergy drugs. Herein, we report for the first time the activity of compounds derived from chalcone scaffolds as inhibitors of normal human basophil degranulation and identified the most active compound with anti-PI3K delta properties that was investigated in preclinical models. Compound 18, namely 1-[2-hydroxy-4,6-dimethoxy-3-(N-methylpiperidin-4-yl)pheny1]-3(2,4,6-trimethoxypheny1)-prop-2-en-1-one, was found to inhibit normal human basophil degranulation in a dose-dependent manner. In a murine model of ovalbumin-induced asthma, compound 18 was shown to reduce expiratory pressure while its impact on the inflammatory infiltrate in alveolar lavage and total lung was dependent on the route of administration. In a DNFB-induced model of atopic dermatitis compound 18 administered systemically proved to be as potent as topical betamethasone. These results support the anti-atopic and allergic properties of the title compound and warrant further clinical development. (C) 2018 Elsevier Masson SAS

International audience; Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated.

Purpose: Multiple myeloma is a clonal plasma cell disorder in which growth and proliferation are linked to a variety of growth factors, including insulin-like growth factor type I (IGF-I). Bortezomib, the first-in-class proteasome inhibitor, has displayed significant antitumor activity in multiple myeloma. Experimental Design: We analyzed the impact of IGF-I combined with proteasome inhibitors on multiple myeloma cell lines in vivo and in vitro as well as on fresh human myeloma cells. Results: Our study shows that IGF-I enhances the cytotoxic effect of proteasome inhibitors against myeloma cells. The effect of bortezomib on the content of proapoptotic proteins such as Bax, Bad, Bak, and BimS and antiapoptotic proteins such as Bcl-2, Bcl-XL, XIAP, Bfl-1, and survivin was enhanced by IGF-I. The addition of IGF-I to bortezomib had a minor effect on NF-κB signaling in MM.1S cells while strongly enhancing reticulum stress. This resulted in an unfolded protein response (UPR), which was required for the potentiating effect of IGF-I on bortezomib cytotoxicity as shown by siRNA-mediated inhibition of GADD153 expression. Conclusions: These results suggest that the high baseline level of protein synthesis in myeloma can be exploited therapeutically by combining proteasome inhibitors with IGF-I, which possesses a “priming” effect on myeloma cells for this family of compounds. Clin Cancer Res; 19(13); 3556–66. ©2013 AACR.

Microtubules are involved in a variety of essential cell functions. Their role during mitosis has made them a target for anti-cancer drugs. However development of resistance has limited their use. It has been established that enhanced beta3-tubulin expression is correlated with reduced response to antimicrotubule agent-based chemotherapy or worse outcome in a variety of tumor settings. However little is known regarding the regulation of beta3-tubulin expression. We investigated the regulatory mechanisms of expression of beta3-tubulin in the MCF-7 cell line, a model of hormone-dependent breast cancer. Exposure of MCF-7 cells to estradiol was found to induce beta3-tubulin mRNA as well as beta3-tubulin protein expression. Conversely, we did not observe induction of beta3-tubulin mRNA by estradiol in MDA-MB-231 cells which are negative for the estrogen receptor (ER). In order to determine whether beta3-tubulin up-regulation is mediated through the ER pathway, MCF-7 cells were exposed to two ER modulators. Exposure to tamoxifen, a selective estrogen receptor modulator, completely abolished the beta3-tubulin mRNA induction due to estradiol in MCF-7 cells. This result was confirmed with fulvestrant, a pure antagonist of ER. These results demonstrate that the effect of estradiol on beta3-tubulin transcription is mediated through an ER dependent pathway.

In mammalian cells, ADP ribosylation factor like 2 (Arl2) has been shown to form a complex with tubulin binding cofactor D (TBC-D) and the tumor suppressor protein phosphatase 2A (PP2A). We have previously shown that alterations in Arl2 protein content were associated with corresponding modifications of the tumor suppressor PP2Ac protein content in breast cancer cells. Here, we show that modified Arl2 expression level influences sensitivity to various anticancer compounds such as taxol, navelbine, gemcitabine and doxorubicin in MCF7 derived cell lines. Modifications of Arl2 expression levels were also associated with an altered phosphorylation status and/or cellular sublocalization of certain PP2A targets such as p53, a key mediator of chemotherapy-induced apoptosis. A decreased level of Arl2 expression was associated with an increase of phospho-ser15-p53, a form which was found to be preferentially bound to microtubules. Assays using okadaic and cantharidic acid, two different PP2A inhibitors, showed an increase in microtubule-bound phospho-p53 and reduced sensitivity to chemotherapy. Our results suggest that Arl2 could, via PP2A, influence p53 phosphorylation status. Certain forms of phosphorylated p53 demonstrating increased binding to microtubules appear to be less prone to nuclear translocation after exposure to chemotherapeutic agents, thereby possibly contributing to reduced chemosensitivity.

Therapeutic mAbs exert antitumor activity through various mechanisms, including apoptotic signalization, complement-dependent cytotoxicity, and antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis (ADCP). G-CSF and GM-CSF have been reported to increase the activity of antibodies in preclinical models and in clinical trials. To determine the potential role of pegfilgrastim as an enhancer of anticancer antibodies, we performed a comparative study of filgrastim and pegfilgrastim. We found that pegfilgrastim was significantly more potent than filgrastim in murine xenograft models treated with mAbs. This was observed with rituximab in CD20+ models and with trastuzumab in HER2+ models. Stimulation with pegfilgrastim was associated with significant enhancement of leukocyte content in spleen as well as mobilization of activated monocytes/granulocytes from the spleen to the tumor bed. These results suggest that pegfilgrastim could constitute a potent adjuvant for immunotherapy with mAbs possessing ADCC/ADCP properties. Mol Cancer Ther; 15(6); 1238–47. ©2016 AACR.

A simple route for improving the potency of progesterone as a modulator of P-gp-mediated multidrug resistance was established by esterification or etherification of hydroxylated 5α/β-pregnane-3,20-dione or 5β-cholan-3-one precursors. X-ray crystallography of representative 7α-, 11α-, and 17α-(2'R/S)-O-tetrahydropyranyl ether diastereoisomers revealed different combinations of axial-equatorial configurations of the anomeric oxygen. Substantial stimulation of accumulation and chemosensitization was observed on K562/R7 erythroleukemia cells resistant to doxorubicin, especially using 7α,11α-O-disubstituted derivatives of 5α/β-pregnane-3,20-dione, among which the 5β-H-7α-benzoyloxy-11α-(2'R)-O-tetrahydropyranyl ether 22a revealed promising properties (accumulation index 2.9, IC50 0.5 μM versus 1.2 and 10.6 μM for progesterone), slightly overcoming those of verapamil and cyclosporin A. Several 7α,12α-O-disubstituted derivatives of 5β-cholan-3-one proved even more active, especially the 7α-O-methoxymethyl-12α-benzoate 56 (accumulation index 3.8, IC50 0.2 μM). The panel of modulating effects from different O-substitutions at a same position suggests a structural influence of the substituent completing a simple protection against stimulating effects of hydroxyl groups on P-gp-mediated transport.

Summary Nucleotide excision repair (NER) is involved in the repair of DNA damage caused by platinum derivatives and has been shown to decrease the cytotoxic activity of these drugs. Because protein–protein interactions are essential for NER activity, we transfected human cancer cell lines (A549 and HCT116) with plasmids coding the amino acid sequences corresponding to the interacting domains between excision repair cross-complementation group 1 (ERCC1) and xeroderma pigmentosum, complementation group A (XPA), as well as ERCC1 and xeroderma pigmentosum, complementation group F (XPF), all NER proteins. Using the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and annexin V staining, we showed that transfected A549 cells were sensitized 1.2–2.2-fold to carboplatin and that transfected HCT116 cells were sensitized 1.4–5.4-fold to oxaliplatin in vitro. In addition, transfected cells exhibited modified in vivo sensitivity to the same drugs. Finally, in particular cell models of the interaction between ERCC1 and XPF, DNA repair was decreased, as evidenced by increased phosphorylation of the histone 2AX after exposure to mitomycin C, and genomic instability was increased, as determined by comparative genomic hybridization studies. The results indicate that the interacting peptides act as dominant negatives and decrease NER activity through inhibition of protein–protein interactions.

International audience; The benefit of cancer chemotherapy based on alkylating agents is limited because of the action of DNA repair enzymes, which mitigate the damage induced by these agents. The interaction between the proteins ERCC1 and XPF involves two major components of the nucleotide excision repair pathway. Here, novel inhibitors of this interaction were identified by virtual screening based on available structures with use of the National Cancer Institute diversity set and a panel of DrugBank small molecules. Subsequently, experimental validation of the in silico screening was undertaken. Top hits were evaluated on A549 and HCT116 cancer cells. In particular, the compound labeled NSC 130813 [4-[(6-chloro-2-methoxy-9-acridinyl)amino]-2-[(4-methyl-1-piperazi nyl) methyl]] was shown to act synergistically with cisplatin and mitomycin C; to increase UVC-mediated cytotoxicity; to modify DNA repair as indicated by the staining of phosphorylated H2AX; and to disrupt interaction between ERCC1 and XPF in cells. In addition, using the Biacore technique, we showed that this compound interacts with the domain of XPF responsible for interaction with ERCC1. This study shows that small molecules targeting the protein-protein interaction of ERCC1 and XPF can be developed to enhance the effects of alkylating agents on cancer cells.

Bivalent ligands were designed on the basis of the described close proximity of the ATP-site and the putative steroid-binding site of P-glycoprotein (ABCB1). The syntheses of 19 progesterone–adenine hybrids are described. Their abilities to inhibit P-glycoprotein-mediated daunorubicin efflux in K562/R7 human leukemic cells overexpressing P-glycoprotein were evaluated versus progesterone. The hybrid with a hexamethylene linker chain showed the best inhibitory potency. The efficiency of these progesterone-adenine hybrids depends on two main factors: (i) the nature of the linker and (ii) its attachment point on the steroid skeleton.

Tubulin binding cofactor C (TBCC) is essential for the proper folding of α- and β-tubulins into microtubule polymerizable heterodimers. Because microtubules are considered major targets in the treatment of breast cancer, we investigated the influence of TBCC silencing on tubulin pools, microtubule dynamics, and cell cycle distribution of breast cancer cells by developing a variant MCF7 cells with reduced content of TBCC (MC−). MC− cells displayed decreased content in nonpolymerizable tubulins and increased content of polymerizable/microtubule tubulins when compared with control MP6 cells. Microtubules in MC− cells showed stronger dynamics than those of MP6 cells. MC− cells proliferated faster than MP6 cells and showed an altered cell cycle distribution, with a higher percentage in S-phase of the cell cycle. Consequently, MC− cells presented higher sensitivity to the S-phase–targeting agent gemcitabine than MP6 cells in vitro. Although the complete duration of mitosis was shorter in MC− cells and their microtubule dynamics was enhanced, the percentage of cells in G2-M phase was not altered nor was there any difference in sensitivity to antimicrotubule-targeting agents when compared with MP6 cells. Xenografts derived from TBCC variants displayed significantly enhanced tumor growth in vivo and increased sensitivity to gemcitabine in comparison to controls. These results are the first to suggest that proteins involved in the proper folding of cytoskeletal components may have an important influence on the cell cycle distribution, proliferation, and chemosensitivity of tumor cells. Mol Cancer Ther; 10(2); 303–12. ©2011 AACR.

Background Microtubules are considered major therapeutic targets in patients with breast cancer. In spite of their essential role in biological functions including cell motility, cell division and intracellular transport, microtubules have not yet been considered as critical actors influencing tumor cell aggressivity. To evaluate the impact of microtubule mass and dynamics on the phenotype and sensitivity of breast cancer cells, we have targeted tubulin binding cofactor C (TBCC), a crucial protein for the proper folding of α and β tubulins into polymerization-competent tubulin heterodimers. Methods We developed variants of human breast cancer cells with increased content of TBCC. Analysis of proliferation, cell cycle distribution and mitotic durations were assayed to investigate the influence of TBCC on the cell phenotype. In vivo growth of tumors was monitored in mice xenografted with breast cancer cells. The microtubule dynamics and the different fractions of tubulins were studied by time-lapse microscopy and lysate fractionation, respectively. In vitro sensitivity to antimicrotubule agents was studied by flow cytometry. In vivo chemosensitivity was assayed by treatment of mice implanted with tumor cells. Results TBCC overexpression influenced tubulin fraction distribution, with higher content of nonpolymerizable tubulins and lower content of polymerizable dimers and microtubules. Microtubule dynamicity was reduced in cells overexpressing TBCC. Cell cycle distribution was altered in cells containing larger amounts of TBCC with higher percentage of cells in G2-M phase and lower percentage in S-phase, along with slower passage into mitosis. While increased content of TBCC had little effect on cell proliferation in vitro, we observed a significant delay in tumor growth with respect to controls when TBCC overexpressing cells were implanted as xenografts in vivo. TBCC overexpressing variants displayed enhanced sensitivity to antimicrotubule agents both in vitro and in xenografts. Conclusion These results underline the essential role of fine tuned regulation of tubulin content in tumor cells and the major impact of dysregulation of tubulin dimer content on tumor cell phenotype and response to chemotherapy. A better understanding of how the microtubule cytoskeleton is dysregulated in cancer cells would greatly contribute to a better understanding of tumor cell biology and characterisation of resistant phenotypes.

Although the correlation between beta3-tubulin expression level in tumors has been well correlated with clinical outcome in patients receiving microtubule-targeted agents, the regulation of this protein remains poorly understood. Recently, new insight of regulatory processes was offered with the cloning of the gene promoter. In this study beta3-tubulin gene expression was induced in response to exposure to various antimicrotubule agents such as vinorelbine and paclitaxel. The exploration of the beta3-tubulin gene promoter by successive deletions followed by site-directed mutagenesis led to the localization of a vinorelbine-responsive element containing an activator-protein 1 (AP-1) site. Among the various antimicrotubule agents tested, it appeared that the implicated AP-1 site was activated only after exposure to vinorelbine. This study confirms the inducible nature of the beta3-tubulin gene promoter.

A series of 59 chalcones was prepared and evaluated for the antimitotic effect against K562 leukemia cells. The most active chalcones were evaluated for their antiproliferative activity against a panel of 11 human and murine cell cancer lines. We found that three chalcones were of great interest as potential antimitotic drugs. In vivo safety studies conducted on one of the most active chalcones revealed that the compound was safe, allowing further in vivo antitumor evaluation.

ADP ribosylation factor like 2 (Arl2) protein is involved in the folding of tubulin peptides. Variants of the human adenocarcinoma line MCF7 cells with increased or reduced content of Arl2 protein were produced and characterized. Western blot analysis performed after separation of the different fractions of tubulins showed that the content in polymerizable soluble heterodimers was significantly increased in cells with the highest Arl2 expression level (MA+) and reduced in cells with the lowest Arl2 expression level (MA-) in comparison to control cells (MP). Microtubule dynamic instability, measured after microinjection of rhodamine-labelled tubulin in living cells, was significantly enhanced in MA+ cells and reduced in MA- cells. These alterations involved modifications of the microtubule growth and shortening rates, duration of attenuation phases, percentage of time spent in each phase (growth, shortening and attenuation) and catastrophe frequency. We also observed modifications in the expression level of the tumor suppressor protein phosphatase 2Ac, which has been shown to form a complex with Arl2. Finally, cell cycle progression was modified in these cells, particularly in regard to duration of telophase. In summary, alterations in Arl2 protein content were found to be associated with modifications in tubulin pools, microtubule dynamics as well as cell cycle progression.

A set of 32 natural and synthetic coumarins were tested in order to evaluate their activity on human leukemic cells (K562/R7) overexpressing P-glycoprotein (P-gp). Their ability to reduce the P-gp-mediated drug efflux of daunorubicin out of cells was evaluated at 10 microM. Four natural compounds, previously isolated from Calophyllum dispar (Clusiaceae) and substituted by a common alpha-(hydroxyisopropyl)dihydrofuran moiety, exhibited a significant inhibitory effect on P-gp when compared to the positive control cyclosporin A. A 3D-quantitative structure-activity relationship (3D-QSAR) analysis of the coumarins was performed using the biological results obtained by comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) of P-gp. Results showed a favorable electrostatic and steric volume, like the alpha-(hydroxyisopropyl)dihydrofuran moiety, beside C(5)-C(6) or C(7)-C(8) positions. In addition, the analysis revealed an important hydrophobic, neutral charge group, like phenyl, in position C(4) on the coumarinic ring.