Background

Oncogenic MAP kinase pathway activating mutations have been shown to drive immune suppression in melanoma and colorectal cancers. In this study, we explored whether oncogenic EGFR mutations play an analogous role in non-small cell lung cancer (NSCLC), and whether EGFRi can relieve tumor-associated immune suppression.

Methods

Lung cancer cell lines expressing wild-type or mutated EGFR were treated with EGFRi, followed by collection of RNA and cell supernatants. In parallel, tumor biopsies from NSCLC patients receiving a personalized peptide vaccine with or without concurrent EGFRi treatment were obtained. RNAseq-based transcriptome profiles of tumor cell lines and patient tumor biopsies were compared to assess common gene signatures driven by EGFRi. Gene expression changes were confirmed at the protein level using Western blot, flow cytometry, and cytokine/chemokine Luminex. The impact of EGFRi on T-cell migration and tumor cell recognition by antigen-specific CD8+ T cells was also assessed.

Results

In addition to downregulating genes associated with cell proliferation, apoptosis and survival, EGFRi increased the transcription of genes associated with TNFa and TRAIL signaling, and antigen presentation. HLA class I protein upregulation was confirmed and correlated with increased recognition of tumor cells by cytotoxic T cells. Several chemokines and cytokines were up- or down-regulated following EGFRi treatment, and Luminex analysis confirmed changes to 10 of them. Migration assays demonstrated that chemotaxis of T cells towards EGFR-mutant cell supernatants increased in an EGFRi dose-dependent manner. Transcriptome profiling of tumor biopsies revealed similar gene expression changes in on-EGFRi treatment tumor samples. Furthermore, increased tumor immune cell infiltration observed in EGFRi-treated patient tumors was consistent with the upregulation of EGFRi signature chemokines at the tumor site.

Conclusions

These results provide evidence that EGFRi has the capacity to facilitate modulation of the tumor microenvironment to favor immune cell infiltration and promote T-cell mediated antitumor immunity.

Legal entity responsible for the study

The University of Texas MD Anderson Cancer Center.

Funding

Tianjin HengJia Biotechnology Development Co., Ltd.

Disclosure

F. Li: Shareholder/Stockholder/Stock options: Tianjin HengJia Biotechnology Development Co., Ltd.. G. Lizee: Advisory/Consultancy: HengJia Neoantigen Biotechnology (Tianjin) Co., Ltd.. P. Hwu: Advisory/Consultancy: Dragonfly Therapeutics; Advisory/Consultancy: GlaxoSmithKline; Advisory/Consultancy, Shareholder/Stockholder/Stock options: Immatics; Advisory / Consultancy: Sanofi; Research grant/Funding (institution): Genentech. L. Deng: Full/Part-time employment: Tianjin HengJia Biotechnology Development Co., Ltd.. Q. Zou: Full/Part-time employment: Tianjin HengJia Biotechnology Development Co., Ltd.. Y. Wang: Full/Part-time employment: Tianjin HengJia Biotechnology Development Co., Ltd.. R. Ataullakhanov: Full/Part-time employment: Bostongene. Llc. Bagaev: Full/Part-time employment: Bostongene. Llc. N. Kotlov: Full/Part-time employment: Bostongene. Llc. V. Svekolkin: Full/Part-time employment: Bostongene. Llc. N. Miheecheva: Full/Part-time employment: Bostongene. Llc. F. Frenkel: Full/Part-time employment: Bostongene. Llc. All other authors have declared no conflicts of interest.

Background

MLH1 knock out mice develop mismatch repair deficient (MMR-D) neoplasias spontaneously and reflect the diverse clinical presentation of MMR-D-driven carcinogenesis in men. The tumor spectrum includes a high prevalence of early Non-Hodgkin T cell lymphomas (NHL), lymphoid skin lesions as well as later developing epithelial tumors of the gastrointestinal tract (GIT).

Methods

Using whole-exome sequencing on MLH1^-/- tumors (2x GIT, 1x splenic NHL, 1x skin lymphoma) as well as GIT-derived cell lines (n = 2), we focused on identification of (I) shared and (II) mutually exclusive mutations and described the processes of ongoing mutational events in tumor-derived cultures.

Results

MLH1^-/- tumors show high tumor mutational burden with 3/4 primary tumor samples even being ultra-hypermutated (> 100 mut/MB). Missense mutations were more frequent than nonsense mutations, base changes were mainly due to transitions (C>T; A>G). The resulting mutational landscape was heterogeneous and in accordance with the human counterpart, MLH1^-/- tumors frequently harbor mutations in PIK3CA, EGFR, KRAS, and/or ERBB3. Of note, only a few shared mutations were detectable among different tumor entities, among them were ARID1A and IDH2. Mutations in classical tumor suppressor genes SMAD4 and POLE were mutually exclusive in lymphomas, most likely contributing to a more aggressive in vivo phenotype. Comparing the mutational profile of selected tumors and their corresponding cell line revealed continuous increased numbers of somatic gene mutations. The same was true for coding microsatellite mutations in MMR-D target genes. Partial overlap was detectable, yet recognizing shared antigens. Two promising candidates are AKT3, a RAC-gamma serine/threonine-protein kinase and the endonuclease ERCC5 (Excision Repair 5), involved in DNA excision repair.

Conclusions

The present study is the first reporting results of a comparison between different spontaneously developing tumors as models for MMR-D driven tumorigenesis. Additionally to identifying ARID1A as causative mutation hotspot, this comprehensive characterization of the mutational landscape may be a good starting point to predict antigens for vaccination approaches.

Legal entity responsible for the study

The authors.

Funding

german research foundation (DFG); grant number: MA5799/2-1.

Disclosure

All authors have declared no conflicts of interest.

Background

Triple-negative breast cancers (TNBCs) are very aggressive and lethal forms of breast cancer with no effective targeted therapy. TNBCs spontaneously metastasize to distant organs including lungs, bone, and brain. Neo-adjuvant chemotherapies and radiotherapy remains a mainstay of treatment with only 25-30% of TNBC patients responding. Thus, there is an unmet clinical need to develop novel therapeutic strategies for TNBCs.

Methods

We tested the anti-cancer activity of auranofin monotherapy on primary tumor growth and metastasis in vitro and in vivo. Further, using in vitro analysis we conceptualized the PD-L1 dependent resistance against auranofin which we validated in vivo using syngeneic TNBC model.

Results

Expression of the thioredoxin pathway genes is significantly upregulated in TNBC patients compared to non-TNBC patients and correlated with adverse survival outcomes. Treatment with auranofin, an FDA-approved thioredoxin reductase inhibitor caused specific cell death and impaired the growth of TNBC grown as spheroids in 3D culture. Further, auranofin treatment exerted a significant in vivo anti-tumor activity in multiple TNBC models including syngeneic 4T1.2 model, human MDA-MB-231 xenograft, and PDX model by inhibiting thioredoxin redox activity. Auranofin also significantly inhibited the invasion potential of TNBC cells in vitro and significantly inhibited lung metastasis in 4T1.2 syngeneic model in vivo by reducing the expression of various EMT markers. We for the first time showed that auranofin increased CD8^+Ve T-cells tumor infiltration in vivo and upregulated immune checkpoint PD-L1 expression in ERK1/2-MYC-dependent manner. Moreover, combination of auranofin with anti-PD-L1 monoclonal antibody synergistically impaired the growth of 4T1.2 primary tumor.

Conclusions

Our data provides a novel therapeutic strategy using auranofin in combination with anti-PD-L1 antibody for TNBCs and warrants further clinical investigation for TNBC patients. Since the success rate of anti-PD-L1 therapy is very low in TNBC patients, our data provides a novel strategy to use auranofin with anti-PD-L1 therapy to that may enhance the efficacy of immune checkpoint therapy in TNBC patients.

Legal entity responsible for the study

The authors.

Funding

Cure Cancer Australia & Can Too Foundation Project grant [ID 1147230] to Prahlad Raninga National Health & Medical Research Council (NH&MRC) Program Grant [ID 1017028] to Kum Kum Khanna Perpetual IMPACT Philanthropy project grant [IPAP201602001] to Kum Kum Khanna and Murugan Kalimutho.

Disclosure

All authors have declared no conflicts of interest.

Background

The role of radiomics against clinical and histologic standard has been repeatedly demonstrated, although the integration of high-throughput imaging into a multidimensional prediction model is still in its early dawn. Thus, the aim of the present study was to determine whether CT-derived radiomic features (CT-RFs) might intercept the landscaped arrangement of the tumor immune microenvironment (TIME), offering a novel non-invasive assessment of prognostic factors in NSCLC.

Methods

A cohort of 100 (70 training and 30 validation) surgically resected NSCLC was investigated. TIME was classified according to PD-L1 and Tumor Infiltrating Lymphocytes (TILs) levels and further defined as hot, intermediate or cold by the relative contribution of effector and suppressor phenotypes. Extracted CT-RFs were correlated to TIME profiles and ROC curves were used to test the accuracy of the radiomic predictor. The impact of integrated radio-immune parameters on clinical outcome was estimated by Kaplan Meier method.

Results

Patient-specific tissue immune profiles were reflected by a strong correlation between CT-RFs and TIME parameters (U-Mann Whitney Test). Cluster Tendency and GrayLevelNonUniformity were upregulated in PD-L1^high and TILs rich tumors (p < 0.01), while Skewness and Coarseness were correlated to PD-L1^low and TILs poor (p < 005) samples, respectively. Among 13 CT extracted features distinctive of hot TIME (hCT-RFs), the most significantly upregulated were Energy, Busyness, and Entropy (p < 0.01), underlining the heterogeneous nature of inflamed NSCLC. Conversely, cold TIME-related features (cCT-RFs) showed a restricted variability being essentially confined to LowGrayLevelEmphasis (p < 0.001). When hCT-RFs and cCT-RFs were entered together in a multivariate logistic regression model, a highly specific and sensitive radiomic predictor of hot (1.00 AUC, p < 0.001) and cold (0.92 AUC, p = 0.001) TIME was revealed. Strikingly, a progressive decrease in OS and DFS (Kaplan Meier, p < 0.001) was documented, respectively, in hot, intermediate and cold NSCLC as defined by the radio-immune model.

Conclusions

Specific TIME profiles inscribe radiomic features resulting in a radio-immune signature with prognostic impact on NSCLC.

Legal entity responsible for the study

University of Parma.

Funding

University of Parma.

Disclosure

All authors have declared no conflicts of interest.

Background

Tumor genome sequencing is becoming widely available in the clinical setting. However, the interpretation of tumor somatic variants remains an important challenge to implement precision cancer medicine.

Methods

Here, we present IntOGen, a platform aimed for tumor genome interpretation. The first component of the platform is an automatic pipeline for cancer driver gene identification across cohorts. This pipeline implements several bioinformatics methods to detect driver genes, on the basis of signals of positive selection in their mutational pattern across tumor cohorts. It also annotates several oncogenomic features that shed light onto the role of driver genes in the development of each malignancy. The second component of the platform leverages these features to identify the somatic mutations that drive the tumor of each patient providing additional information about the possible effect of driver mutations on treatment response.

Results

We have applied the framework to more than 50,000 tumors across more than 60 tumors types creating the largest repository of tumor genetic driver events. This comprehensive oncogenomics repository is the third component of IntOGen.

Conclusions

All the results and framework are available online to the cancer genomics research community at www.intogen.org, which we envision can support a broad range of oncology use cases.

Legal entity responsible for the study

Institute for Research in Biomedicine (IRB, Barcelona).

Funding

Ministerio de Educación y Ciencia de España (SAF-2015-R-66084 and RTI2018-094095-B-I00) Instituto Nacional de Bioinformática (INB) - PT17/0009/0013.

Disclosure

All authors have declared no conflicts of interest.

Background

Standard CUP therapy has not changed for decades. CUPISCO (NCT03498521) is an ongoing randomised prospective trial using CGP to assign CUP pts to individualised targeted or immunotherapy arms (molecularly guided therapy; MGT).

Methods

Archival tissue from 303 centrally reviewed undifferentiated- and adeno-CUP cases in the FoundationCore^™ database underwent hybrid capture-based CGP (FoundationOne^® CDx). Microsatellite instability (MSI), tumour mutational burden (TMB) and genomic loss of heterozygosity (gLOH) were calculated (Frampton Nat Biotechnol 2013; Swisher Lancet Oncol; Chambers Genome Med 2017). PD-L1 expression was measured by DAKO 22C3 immunohistochemistry. Pts were classified by whether CGP results could have informed assignment to CUPISCO arms; TTF-1+, CK7–/CK20+/CDX2+ or TMPRSS2:ERG+ cases were excluded.

Results

The sex ratio was 1:1; median age was 67 yrs (range 22–89+). CGP revealed 96 pts (32%) matched to a CUPISCO arm (Table).Table:

1983PD_PR

Conclusions

32% of CUP pts would have been potentially eligible for MGT in CUPISCO. Future studies including additional biomarkers, such as PD-L1–positivity and gLOH, may identify a greater proportion of CUP pts potentially benefitting from individualised treatment.

Editorial acknowledgement

Support for third-party editing assistance for this abstract, furnished by Daniel Clyde, PhD, of Health Interactions, was provided by F. Hoffmann-La Roche Ltd, Basel, Switzerland.

Legal entity responsible for the study

F. Hoffmann-La Roche Ltd.

Funding

F. Hoffmann-La Roche Ltd.

Disclosure

J.S. Ross: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine, Inc.; Non-remunerated activity/ies, Support for third-party editing assistance: F. Hoffmann-La Roche Ltd. E.S. Sokol: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine, Inc; Non-remunerated activity/ies, Support for third-party editing assistance: F. Hoffmann-La Roche Ltd. H. Moch: Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: F. Hoffmann-La Roche Ltd ; Advisory / Consultancy, Travel / Accommodation / Expenses: Definiens AG; Non-remunerated activity/ies, Support for third-party editing assistance: F. Hoffmann-La Roche Ltd. L. Mileshkin: Travel / Accommodation / Expenses: F. Hoffmann-La Roche Ltd; Travel / Accommodation / Expenses: Beigene; Non-remunerated activity/ies, Support for third-party editing assistance: F. Hoffmann-La Roche Ltd. G. Baciarello: Honoraria (self), Travel / Accommodation / Expenses: F. Hoffmann-La Roche Ltd; Honoraria (self), Travel / Accommodation / Expenses: Astellas; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Janssen; Honoraria (self), Travel / Accommodation / Expenses: Ipsen; Honoraria (self): Amgen; Travel / Accommodation / Expenses: AstraZeneca; Non-remunerated activity/ies, Support for third-party editing assistance: F. Hoffmann-La Roche Ltd; Advisory / Consultancy, Travel / Accommodation / Expenses: Sanofi. F. Losa: Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution), Travel / Accommodation / Expenses: F. Hoffmann-La Roche Ltd; Advisory / Consultancy, Speaker Bureau / Expert testimony: Sanofi; Advisory / Consultancy, Research grant / Funding (institution): Amgen; Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Merck; Advisory / Consultancy: Servier; Non-remunerated activity/ies, Support for third-party editing assistance: F. Hoffmann-La Roche Ltd. A. Beringer: Full / Part-time employment, Non-remunerated activity/ies, Support for third-party editing assistance: F. Hoffmann-La Roche Ltd. M. Thomas: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment, Non-remunerated activity/ies, Support for third-party editing assistance: F. Hoffmann-La Roche Ltd. S. Foser: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment, Non-remunerated activity/ies, Support for third-party editing assistance: F. Hoffmann-La Roche Ltd. J. Elvin: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine, Inc; Non-remunerated activity/ies, Support for third-party editing assistance: F. Hoffmann-La Roche Ltd. N. Danziger: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine, Inc; Non-remunerated activity/ies, Support for third-party editing assistance: F. Hoffmann-La Roche Ltd. N. Ngo: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine, Inc; Non-remunerated activity/ies, Support for third-party editing assistance: F. Hoffmann-La Roche Ltd. J.Y. Tse: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine, Inc; Non-remunerated activity/ies, Support for third-party editing assistance: F. Hoffmann-La Roche Ltd. K. Killian: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine, Inc; Full / Part-time employment, Special Volunteer: National Cancer Institute; Shareholder / Stockholder / Stock options, Non-remunerated activity/ies, Support for third-party editing assistance: F. Hoffmann-La Roche Ltd. D.X. Jin: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine, Inc; Shareholder / Stockholder / Stock options, Non-remunerated activity/ies, Support for third-party editing assistance: F. Hoffmann-La Roche Ltd. L.M. Gay: Full / Part-time employment, Current: Ellem Consulting; Non-remunerated activity/ies, Support for third-party editing assistance: F. Hoffmann-La Roche Ltd; Full / Part-time employment, Previous: Foundation Medicine, Inc. A. Krämer: Honoraria (self), Honoraria (institution), Advisory / Consultancy, Travel / Accommodation / Expenses, Non-remunerated activity/ies, Support for third-party editing assistance: F. Hoffmann-La Roche Ltd; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Daiichi Sankyo; Honoraria (self), Advisory / Consultancy: Abbvie; Honoraria (institution), Research grant / Funding (institution): Bayer; Advisory / Consultancy: BMS; Travel / Accommodation / Expenses: Celgene; Research grant / Funding (institution): Merck.

Background

Different cancers subtypes can often be effectively treated with similar Rx classes, i.e., with PARPi in the case of BRCA mutations or with anti-PD1/PDL1 inhibitors for patients with mismatch repair deficiencies. Using the Epic Sciences Functional Cell Profiling (FCP) platform utilizing high resolution digital pathology and machine learning, we analyzed ∼100,000 single CTCs from multiple indications to index metastatic cancers, with the longer-term hypothesis that sub-classification of CTCs may help define therapeutic decisions across cancer types.

Methods

92,300 CTCs underwent FCP analysis (single cell digital pathology features of cellular and sub-cellular morphometrics) were collected from cancer patients comprising Prostate (1641 pts, 70,747 CTCs), Breast (268 pts, 8,718 CTCs), NSCLC (110 pts, 1884 CTCs), SCLC (141 pts, 8,872 CTCs) and Bladder (65 pts, 2079 CTCs). An equal number of CTCs from each indication was sampled to create a balanced cohort for training. K-means clustering was applied on the training set and an optimized number of clusters were determined using the elbow approach. After defining the clusters on the training set, the cluster centers were extracted from k-means, and used to train a k-Nearest Neighbor (k-NN) classifier to predict the cluster assignment for the remaining CTCs designated as the test set.

Results

The optimized number of clusters was 9. Breast cancer CTCs were more enriched with higher CK expressing CTCs (c1), while SCLC and some of mCRPC shared similar small cell like features (c5). Cluster subtypes and characteristics are listed.Table:

1984PD

Conclusions

Heterogeneous CTC phenotypic subtypes were observed across multiple indications. Each indication harbored subtype heterogeneity and shared clusters with other cancers. Patient cluster subtype analysis for prognosis and therapy benefit as well as linkage of CTC subtype genotypes to patient survival by cancer type is ongoing.

Legal entity responsible for the study

The authors.

Funding

Epic Sciences.

Disclosure

A. Jendrisak: Full / Part-time employment: Epic Sciences. R. Sutton: Full / Part-time employment: Epic Sciences. S. Orr: Full / Part-time employment: Epic Sciences. D. Lu: Full / Part-time employment: Epic Sciences. J. Schonhoft: Full / Part-time employment: Epic Sciences. Y. Wang: Full / Part-time employment: Epic Sciences.

Background

Snail is a transcriptional repressor that promotes epithelial-to-mesenchymal transition (EMT). Snail is upregulated in breast cancer and it is associated with chromosomal instability (CIN) and metastasis. The mitotic spindle assembly checkpoint (SAC) is a critical mechanism to prevent CIN. Extensive studies have investigated signaling pathways that involve in protein-protein interactions and post translational modifications. However, it not known how mitosis might be impacted by transcriptional regulation. Due to the fact that Snail is a transcriptional regulator and that Snail is upregulated in breast cancer with CIN, it is critical to understand whether and how abnormally regulated Snail contributes to breast cancer CIN via transcriptional regulation.

Methods

We used siRNA to knockdown Snail in breast caner cell lines to look for a change in SAC. We developed a phosphor-specific antibody to study Aurora-B mediated phosphorylation. Site specific mutagenesis was used to generate phosphorylation site mutants, and exogenous proteins were expressed in cells to look for an effect in SAC and CIN.

Results

We found that Snail is an essential element that is required for the SAC. Genetic depletion of Snail in breast cancer cells by siRNA led to a defect in activation of SAC (measured by accumulation of Histone Serine 10 phosphorylation in response to spindle damaging agents). Interestingly we also found that the Snail protein level was significantly enhanced in the presence of nocodazole. Further, we found that Snail upregulation was through phosphorylation in an Aurora-B dependent manner. We identified a serine site that can be phosphorylated by Aurora-B. Using a phosphor-specific antibody we have developed, we further proved that the phosphorylation event was initiated in mitosis. Functional studies reveal a critical role for Snail phosphorylation in activation of the SAC and prevention of CIN.

Conclusions

1. Snail is required for mitotic spindle checkpoint activation; 2. Snail is phosphorylated by Aurora B in mitosis; 3. Aurora-B medicated Snail phosphorylation is required for optinal activation of the spindle checkpoint and prevention of chromosomal instability in breast cancer.

Legal entity responsible for the study

The authors.

Funding

Tianjin Medical University Cancer Institute and Hospital.

Disclosure

All authors have declared no conflicts of interest.

Background

Aspartame (asparagynal)-β-hydroxylase (ASPH) leads to hepatocellular carcinoma (HCC) progression, but the potential molecular mechanism of ASPH on HCC progression to metastasis is still obscure. Our aim was to explore the correlation between ASPH and HIF1α-mediated mitophagy fueling via glutamine during tumor progression to metastasis in HCC.

Methods

Bioinformatics analysis constructed a composite gene-gene functional interaction network for ASPH regulatory genes to predict potential cellular mechanisms and then verified in vivo and vitro. ASPH+/-expressing stable cell line of HepG2 and Huh7 cell lines were structured by lentivirus encoding the wild-type ASPH open reading frame and ASPH-CRISPR/Cas9 system. 140 cases of HCC patients and a sbcutaneous xenograft HCC tumor model were used to evaluate HCC tumor growth and progression in vivo. The effect of ASPH on mitophagy, glutamine metabolism, angiogenesis and metastasis in HCC were tested via western blot, RT-PCR, immunohistochemical staining, electron microscopy, hematoxylin-eosin (HE) and immunocytofluorescent staining. The HIF1α inhibitor 2-Methoxyestradiol (2-MeOE2) and the serum free cultivation were used to conduct the related rescue experiments in vitro.

Results

In this study, ASPH improved the expression of LC3-II/LC3-I,Atg7, Atg5 and Beclin1 and down-regulated the expression of TOMM20 and P62. Meanwhile, ASPH also significantly increased the expression of GLS1, which reflects the glutamine metabolism level of tumor cells. In addition, ASPH also significantly inhibited the expressions of HIF1α, BNIP3 and NIX proteins in HCC. Furthermore, a significant association between ASPH and CD34 expression was observed in HCC patients (P = 0.024).

Conclusions

Our finding suggests that ASPH fuels glutamine via HIF1α-mediated mitophagy to drive cancer angiogenesis and metastasis in HCC. ASPH may act as a promising therapeutic target in HCC patients.

Legal entity responsible for the study

The authors.

Funding

National Natural Science Foundation of China (No. 81470877) Beijing Natural Science Foundation (No.7192085).

Disclosure

All authors have declared no conflicts of interest.

Background

Long non-coding RNAs (lncRNAs) have been recently identified as key players in several cancer-associated pathways such as metastasis and drug resistance. Emerging evidence indicates that the HORAS5 lncRNA (i.e. linc00161) modulates drug response in different malignancies. In a recently published study, we have shown that HORAS5 promotes the survival of androgen receptor-positive (AR⁺) castration resistant prostate cancer (CRPC) cells. Preliminary data obtained in our lab show that HORAS5 is involved in response to cabazitaxel, in both AR⁺ and AR^- CRPC cells.

Methods

HORAS5 expression was modulated in AR⁺ and AR^- CRPC cells with lentiviral-mediated overexpression and siRNA-based silencing. We measured cell count (Trypan Blue exclusion test) and apoptosis (caspase 3/7) of CRPC cells exposed to clinically achievable concentrations of cabazitaxel. RNA sequencing, RT-qPCR and western blot were used to identify genes regulated by HORAS5 in cabazitaxel-treated cells. We also transfected the cells with antisense oligonucleotides (ASOs), a technology that is currently being tested in clinical trials.

Results

Our results show that HORAS5 overexpression increases cabazitaxel IC₅₀ (p = 0.01), while HORAS5 silencing has an opposite effect (p = 0.003). HORAS5 also inhibits caspase activity upon cabazitaxel treatment (p < 0.0001). RNA sequencing and RT-qPCR revealed that BCL2A1 is the most upregulated transcript in HORAS5 overexpressing cells exposed to cabazitaxel (p < 0.0001), and that BCL2A1 silencing decreases cell count (p < 0.03) and increases caspase activity (p < 0.0001). This evidence suggests that BCL2A1 expression is induced by HORAS5, thereby enhancing CRPC cells resistance to cabazitaxel. Transfection of CRPC cells with HORAS5-targeting ASOs can effectively silence this lncRNA (∼80% KD) and determine a decrease in the IC₅₀ (p = 0.03) in response to cabazitaxel.

Conclusions

Our findings show that HORAS5 mediates cabazitaxel resistance in both AR⁺ and AR^- CRPC cells via regulation of the anti-apoptotic BCL2A1. Experiments with ASOs demonstrate the translational potential of HORAS5 inhibition. We are currently assessing HORAS5 expression in biological fluids from patients exposed (or not) to cabazitaxel.

Legal entity responsible for the study

Francesco Crea.

Funding

The Open University.

Disclosure

All authors have declared no conflicts of interest.