Parental Wild-type Cells Research Articles

Functional Interdependence of Anoctamins May Influence Conclusions from Overexpression Studies.

Anoctamin 6 (ANO6, TMEM16F) is a phospholipid (PL) scramblase that moves PLs between both plasma membrane (PM) leaflets and operates as an ion channel. It plays a role in development and is essential for hemostasis, bone mineralization and immune defense. However, ANO6 has also been shown to regulate cellular Ca2+ signaling and PM compartments, thereby controlling the expression of ion channels such as CFTR. Given these pleiotropic effects, we investigated the functional interdependence of the ubiquitous ANO6 with other commonly co-expressed anoctamins. As most expression studies on anoctamins use HEK293 human embryonic kidney cells, we compared ion currents, PL scrambling and Ca2+ signals induced by the overexpression of anoctamins in HEK293 wild-type parental and ANO6-knockout cells. The data suggest that the endogenous expression of ANO6 significantly affects the results obtained from overexpressed anoctamins, particularly after increasing intracellular Ca2+. Thus, a significant interdependence of anoctamins may influence the interpretation of data obtained from the functional analysis of overexpressed anoctamins.

The mechanistic role of connexin 43 in maturation of hiPSC cardiomyocytes

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): ECR (European research council) and ReNew (the Novo Nordisk Foundation Center for Stem Cell Medicine supported by Novo Nordisk Foundation grants) Introduction Cardiomyocytes (CMs) differentiated from human induced pluripotent stem cells (hiPSCs) are functionally immature and this limits the application of these cells to cardiovascular research. Maturation can be improved by aggregating hiPSC-CMs into three-dimensional (3D) microtissues together with hiPSC-cardiac fibroblasts (hiPSC-CFs) and hiPSC-endothelial cells (hiPSC-ECs). These cardiac microtissues showed improved structural (increase of sarcomere length and organisation) and functional (more mature action potential profile) properties. This effect was only observed when the cardiac microtissues contained hiPSC-CFs. Since connexin 43 (CX43), the most ubiquitous connexin isoform in the heart, was significantly upregulated in hiPSC-CMs from cardiac microtissues, we hypothesise that mechanisms underlying hiPSC-CMs maturation involves the coupling of hiPSC-CMs with hiPSC-CFs through this protein. Purpose The aim of this study is to unravel the role of CX43 in the maturation of hiPSC-CMs in the context of 3D multicellular cardiac tissues. Methods We used the CRISPR/Cas9 technology to generate a CX43 knock-out (KO) hiPSC line. We designed two sgRNAs annealing to exon 2 of GJA1 gene, encoding CX43; indels generated by the non-homologous end joining after the Cas9 double strand break will result in a non-functional protein. Cells were differentiated in vitro into CMs, epicardial cells (EPI) and then CFs and characterized for the expression of cell-specific markers and compared with the wild-type parental control cells. Results and conclusions We screened 31 hiPSC clones and selected those in which both GJA1 alleles contained a deletion. Copy number variation analysis by ddPCR of the flanking regions confirmed that the deletion was specific and confined. The lack of GJA1 expression in the KO clones was confirmed by qPCR, while the lack of the CX43 protein was confirmed by immunofluorescence and Western Blot. This did not affect CX43-KO hiPSCs pluripotency state shown by the presence of pluripotent markers such as NANOG, OCT3/4, SSEA4. Additionally, CX43-KO hiPSCs were able to differentiate into the three germ layers. CX43-KO hiPSCs were successfully differentiated into hiPSC-CMs, -EPI, and -CFs. CX43-KO hiPSC-CMs and controls showed comparable gene expression of cardiac troponin T. Since immunofluorescent staining showed that CX43-KO hiPSC-EPI expressed the epicardial markers WT1 and ZO1, we continued to differentiate them into hiPSC-CFs, which expressed markers such as VIM and COL1A1, similar to the control. Differentiated CX43-KO hiPSC-CMs and CFs will be assembled into cardiac microtissues. Functional and structural assays that include patch-clamp and immunofluorescence will be performed on these microtissue to assess the effect of the CX43 KO on the maturation of hiPSC-CMs. Our results show that the lack of CX43 expression does not impede hiPSCs to differentiate into various cell types originating from the mesoderm germ layer.

Abstract P22: C16orf72/HAPSTR1/TAPR1 Interacts with BRCA1 and Senataxin to Regulate R-loops at Stalled Replication Forks and Confer Resistance to PARP Disruption

Abstract Poly (ADP-ribose) polymerase (PARP) proteins play a crucial role in DNA repair, and their inhibition is toxic in cells with defects in homologous recombination (HR). Whilst this interaction is well-established, other synthetic lethal interactions with PARP1/PARP2 disruption are poorly explored. Hence, we performed a genome-wide CRISPR screen to identify genes which are synthetic lethal with PARP1/PARP2 disruption. We discovered that the uncharacterized C16orf72/HAPSTR1/TAPR1 gene is essential for the survival of PARP1/PARP2 double knock-out cells but not the parental wildtype cells. We further show that C16orf72 is a novel modulator of R-loop and is crucial in facilitating replication fork restart and suppressing DNA damage in response to replication stress. We show that C16orf72 functions in a parallel pathway to PARP1/PARP2 to suppress DNA:RNA hybrids that accumulate at stalled replication forks, and it does this by interacting with BRCA1 and the DNA/RNA helicase Senataxin. Citation Format: Abhishek Bharadwaj Sharma, Muhammad Khairul Ramlee, Joel Kosmin, Martin R. Higgs, Amy Wolstenholme, George E. Ronson, Dylan Jones, Daniel Ebner, Noor Shamkhi, David Sims, Paul W. G. Wijnhoven, Josep Forment, Ian Gibbs-Seymour, Nicholas D. Lakin. C16orf72/HAPSTR1/TAPR1 Interacts with BRCA1 and Senataxin to Regulate R-loops at Stalled Replication Forks and Confer Resistance to PARP Disruption [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P22.

Abstract 6043: Discovery and characterization of a p300-selective degrader with potent anti-tumor activity in CBP mutant cancers

Abstract Although precision medicine strategies for the treatment of human cancers are clinically successful, they have also revealed that only a small fraction of cancers carry actionable alterations in oncogenic drivers. To expand the druggable genome while maintaining an emphasis on genetically targeted therapies, recent efforts have focused on exploiting synthetic lethal relationships. Paralogous protein pairs are particularly compelling targets in this context due to the clear mechanistic basis for the synthetic lethality. As such, functional genomics screens as well as work from Ogiwara et al. have shown compelling rationale for targeting the histone acetylase transferase p300 in the context of CBP-deficient cancer types. We discovered potent heterobifunctional degraders with demonstrated selectivity for p300 over CBP. Our compound exhibits degradation of p300 within 2 hours (DC50 < 10nM, Dmax > 90%) with minimal impact on CBP through 48 h (DC50 >1uM, Dmax = 30%). Utilizing CRISPR-generated isogenic cell lines depleted for either CBP or p300, we observed significant growth inhibition in CBP knockout (KO) cells (gIC50 = 17nM) with minimal impact on growth of p300 KO (gIC50 = >10uM) or parental wild-type cells (gIC50 > 10uM). This translated to downstream pharmacology, where we observed a rapid and potent inhibition of global H3K27 acetylation in the CBP deficient context but a significantly attenuated response in p300 deleted or parental cells. We subsequently employed computational methods to identify and characterize CBP alterations, defining a putative loss-of-function (LoF) phenotype and confirming this sensitivity pharmacologically in cancer cell types harboring these endogenous mutations. Results mirrored what we observed with CBP depletion in these endogenous CBP LoF cell lines, with potent growth inhibition seen in multiple cell lines including LK-2 (gIC50 = 8nM), H1703 (gIC50 = 9nM), and TE-8 cells (gIC50 = 23nM). To confirm the in vitro results in an in vivo setting, we evaluated the response in mice harboring H1703 xenografts. Once daily oral administration of our compound led to almost complete degradation of p300 within H1703 xenograft tumors, resulting in a pronounced inhibition of tumor growth. Lastly, we profiled p300 degradation in human bone marrow derived myeloid progenitor colony-forming assays where we observed a significant reduction in toxicity (IC50 = 3.9uM) compared to a dual CBP/p300 inhibitor (IC50 = 121nM) or degrader (IC50 = 16nM), supporting the hypothesis that a p300-specific mechanism will offer an improved therapeutic index while maintaining anti-tumor efficacy in a biomarker population Taken together, our results suggest that a p300-selective degrader has the potential to serve as an effective therapeutic modality in CBP-mutated cancers. Citation Format: Mike R. Russell, Cassandra L. Lowenstein, Xuqing Zhang, Jeremy Roach, Jianing Song, Rakesh Nagilla, Nathan Kendsersky, Shreyas Joshi, Peter Orth, Matt Tudor, Qiaolin Deng, Clemente Aguilar-Bonavides, Elham Behshad, Sudeep Banjade, Zhihua Sui, Corey Strickland, Larry Jolivette, Helai P. Mohammad. Discovery and characterization of a p300-selective degrader with potent anti-tumor activity in CBP mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6043.

Abstract 2871: Co-targeting asparagine and glutamine metabolism is a viable treatment strategy for PI3K/PTEN mutated cervical cancer

Abstract Purpose Statement: Alterations in the PI3K/AKT signaling pathway are common in cervical cancer. The most commonly observed mutations are in PIK3CA (26%) and PTEN (8%). However, the biological and metabolic implications of acquiring these mutations in the cervical cancer genome are poorly characterized. Materials/Methods: Isogenic cell lines with PIK3CA E545K and PTEN null mutations were generated using Crispr/CAS9. Cell viability and clonogenic cell survival assays were performed in the presence and absence of inhibitors of glutamine and asparagine metabolism. Glucose and glutamine metabolism was evaluated by stable isotope labeling with U13C glucose and13C5 glutamine in presence of glutamine antagonist DON. RNA-seq was employed to evaluate transcriptomic alterations. Results: Introduction of the PIK3CA E545K mutation and PTEN mutation in cervical tumor cells leads to an increase in AKT signaling compared to the parental (wild-type) cells. Notably, RNASeq and mass spectrometric analysis of both the tumor cells and their culture media revealed heightened dependencies on glucose and glutamine in the tumor cells carrying the PIK3CA E545K and PTEN mutations as compared to the wild-type cells. Treatment with glutamine antagonists (DON, JHU-083) and GLS inhibitors (CB-839 and IACS-6274), resulted in reduced cell viability and clonogenic survival in tumor cells with PI3K/PTEN mutation compared to wild-type. Additionally, we observed a metabolic reliance on the amino acid asparagine in tumors with PI3K/PTEN alterations when treated with DON. Asparaginase in combination with DON effectively inhibited the proliferation of human and mouse cell lines derived from primary tumors with PTEN mutations. Furthermore, the incorporation of a glutamine antagonist and asparaginase into conventional chemoirradiation further enhanced the therapeutic efficacy in cervical tumors with PI3K/PTEN alterations which are otherwise treatment resistant. Conclusions: Orthogonal evaluations using glutamine agonists, GLS inhibitors, and mass spectrometric point to a significant role played by PI3K/PTEN mutations in altering cervix tumor cell metabolism towards a more glutamine dependent phenotype. This study suggests a potential strategy for improving the treatment of PI3K-AKT-altered cervical tumors by targeting glutamine and asparagine metabolism in combination with standard therapies. These treatment strategies are currently being evaluated in vivo using a patient derived xenograft model with PIK3CA E545K mutation as well as an immunocompetent cervical tumor model in mice with PTEN deletion. Citation Format: Nishanth Noel Gabriel, Naoshad Muhammad, Kevin Cho, Kay Jayachandran, Jin Zhang, Gary Patti, Julie Schwarz. Co-targeting asparagine and glutamine metabolism is a viable treatment strategy for PI3K/PTEN mutated cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2871.

ARHGAP29 Is Involved in Increased Invasiveness of Tamoxifen-resistant Breast Cancer Cells and its Expression Levels Correlate With Clinical Tumor Parameters of Breast Cancer Patients.

Aggressive breast cancer (BC) cells show high expression of Rho GTPase activating protein 29 (ARHGAP29), a negative regulator of RhoA. In breast cancer cells in which mesenchymal transformation was induced, ARHGAP29 was the only one of 32 GTPase-activating enzymes whose expression increased significantly. Therefore, we investigated whether there is a correlation between expression of ARHGAP29 and tumor progression in BC. Since tamoxifen-resistant BC cells exhibit increased mesenchymal properties and invasiveness, we additionally investigated the relationship between ARHGAP29 and increased invasion rate in tamoxifen resistance. The question arises as to whether ARHGAP29 is a suitable prognostic marker for the progression of BC. Tissue microarrays were used to investigate expression of ARHGAP29 in BC and adjacent normal breast tissues. Knockdown experiments using siRNA were performed to investigate the influence of ARHGAP29 and the possible downstream actors RhoC and pAKT1 on invasive growth of tamoxifen-resistant BC spheroids in vitro. Expression of ARHGAP29 was frequently increased in BC tissues compared to adjacent normal breast tissues. In addition, there was evidence of a correlation between high ARHGAP29 expression and advanced clinical tumor stage. Tamoxifen-resistant BC cells show a significantly higher expression of ARHGAP29 compared to their parental wild-type cells. After knockdown of ARHGAP29 in tamoxifen-resistant BC cells, expression of RhoC was significantly reduced. Further, expression of pAKT1 decreased significantly. Invasive growth of three-dimensional tamoxifen-resistant BC spheroids was reduced after knockdown of ARHGAP29. This could be partially reversed by AKT1 activator SC79. Expression of ARHGAP29 correlates with the clinical tumor parameters of BC patients. In addition, ARHGAP29 is involved in increased invasiveness of tamoxifen-resistant BC cells. ARHGAP29 alone or in combination with its downstream partners RhoC and pAKT1 could be suitable prognostic markers for BC progression.

Truncated O-glycosylation in metastatic triple-negative breast cancer reveals a gene expression signature associated with extracellular matrix and proteolysis

Breast cancer (BC) is the leading cause of death by cancer in women worldwide. Triple-negative (TN) BC constitutes aggressive and highly metastatic tumors associated with shorter overall survival of patients compared to other BC subtypes. The Tn antigen, a glycoconjugated structure resulting from an incomplete O-glycosylation process, is highly expressed in different adenocarcinomas, including BC. It also favors cancer growth, immunoregulation, and metastasis in TNBC. This work describes the differentially expressed genes (DEGs) associated with BC aggressiveness and metastasis in an incomplete O-glycosylated TNBC cell model. We studied the transcriptome of a TNBC model constituted by the metastatic murine 4T1 cell line that overexpresses the Tn antigen due to a mutation in one of the steps of the O-glycosylation pathway. We analyzed and compared the results with the parental wild-type cell line and with a Tn-negative cell clone that was poorly metastatic and less aggressive than the 4T1 parental cell line. To gain insight into the generated expression data, we performed a gene set analysis. Biological processes associated with cancer development and metastasis, immune evasion, and leukocyte recruitment were highly enriched among functional terms of DEGs. Furthermore, different highly O-glycosylated protein-coding genes, such as mmp9, ecm1 and ankyrin-2, were upregulated in 4T1/Tn+ tumor cells. The altered biological processes and DEGs that promote tumor growth, invasion and immunomodulation might explain the aggressive properties of 4T1/Tn+ tumor cells. These results support the hypothesis that incomplete O-glycosylation that leads to the expression of the Tn antigen, which might regulate activity or interaction of different molecules, promotes cancer development and immunoregulation.

Direct, Potent, and TP53-Independent Activity of Sting Agonists Against Acute Myeloid Leukaemia Enhances Venetoclax Efficacy In Vivo

Introduction TP53 mutation occurs in approximately 20% of patients with newly diagnosed acute myeloid leukaemia (AML). Treatment outcomes are universally poor and discovery of new therapies with TP53 independent activity represents an area of very high unmet need. Cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) sense cytoplasmic double-stranded DNA to elicit type I interferon production. Direct pharmacologic activation of STING has been investigated in clinical trials with the goal of enhancing immunogenicity. During apoptosis, activated caspases have been shown to suppress activation of the cGAS/STING pathway to render cell death immunologically silent. Whilst the canonical role of STING is stimulation of an inflammatory response, we now show that direct activation of STING by potent and clinically relevant agonists can induce apoptosis directly in cancerous cells by leveraging the high levels of expressed STING in a variety of blood cancers. Importantly, apoptosis induced by STING agonists may be enhanced through combination with diverse BH3-mimetic drugs in a TP53 independent manner (Diepstraten et al, submitted). In this abstract, we highlight the activity of STING agonists in AML and its potential for clinical testing, especially among patients with TP53 mutated disease. Methods We tested the clinically relevant small molecule STING agonist diABZI (purchased from SYNthesis Med Chem), which is currently in early phase clinical trials (NCT03843359, NCT05424380). diABZI was tested alone or in combination with the BCL-2 specific BH3 mimetic drug venetoclax (purchased from Chemgood). Drug sensitivity assays were performed using human AML derived cell lines and primary bone marrow or peripheral blood mononuclear cells from patients with AML or myelodysplastic syndrome with excess blasts 2 (MDS-EB2). Cells were cultured for 48 hours, and cell death assessed by PI or DAPI staining. In vivo experiments were conducted by transplanting NOD- scidIL2Rgamma null (NSG) mice with MOLM-13 TP53 wildtype (WT) or TP53 knockout(KO) cells. Mice then received either no treatment (control), diABZI alone (1.5 mg/kg intravenously twice a week), venetoclax alone (50 mg/kg orally Monday-Friday), or combination diABZI and venetoclax for two weeks ( n=5 per treatment arm). Survival analysis was performed using the log-rank (Mantel-Cox) test with Bonferroni correction. Results Single agent diABZI was active in 5/6 human AML cell lines tested, including cell lines with TP53 mutation. Isogenic MOLM-13 TP53 KO cells demonstrated the same sensitivity as the TP53 WT parental cells. BAX/BAK KO and STING KO cell lines were resistant to diABZI, confirming that cell death occurred by apoptosis being dependent on BAX/BAK as well as STING. Combination of diABZI and venetoclax resulted in additive killing in MOLM-13 and OCI-AML3 cells (Bliss scores of 7.62 and 8.06, respectively). Ex vivo testing was performed on 18 different primary specimens (16 AML and 2 MDS-EB2; 12/18 cases had high risk genetic or clinical features such TP53 mutation, complex karyotype, secondary disease, or relapse after prior therapy) (Figure 1). Strikingly, diABZI was active as a single agent in 16/18 cases, with marked efficacy in 11 cases (IC 50 <100 nM). The addition of venetoclax enhanced killing of AML cells, especially in cases resistant to STING agonist alone. In vivo, diABZI monotherapy and combination therapy with venetoclax was well tolerated without significant weight loss or hematologic toxicity. Treatment with diABZI prolonged survival in the highly aggressive MOLM-13 xenograft model (Figure 2). In both the TP53 WT and TP53 KO context, single agent diABZI was superior to single agent venetoclax ( p=0.012 and p=0.011, respectively), but survival was most prolonged in mice receiving combination therapy ( p=0.013and p=0.012, respectively). Conclusion Activation of STING can exert direct anti-tumour effects in blood cancers via induction of apoptosis. Human AML cells are highly sensitive to the pro-apoptotic effects of STING agonists. The combination of venetoclax and STING agonist shows marked efficacy in ex vivo and in vivo studies, including in models of TP53 defective AML. Combining STING agonists with venetoclax represents a highly promising and novel therapeutic approach for AML and should be considered for accelerated early phase clinical trials.

Transcriptome-Based Traits of Radioresistant Sublines of Non-Small Cell Lung Cancer Cells.

Radioresistance is a major obstacle for the successful therapy of many cancers, including non-small cell lung cancer (NSCLC). To elucidate the mechanism of radioresistance of NSCLC cells and to identify key molecules conferring radioresistance, the radioresistant subclones of p53 wild-type A549 and p53-deficient H1299 cell cultures were established. The transcriptional changes between parental and radioresistant NSCLC cells were investigated by RNA-seq. In total, expression levels of 36,596 genes were measured. Changes in the activation of intracellular molecular pathways of cells surviving irradiation relative to parental cells were quantified using the Oncobox bioinformatics platform. Following 30 rounds of 2 Gy irradiation, a total of 322 genes were differentially expressed between p53 wild-type radioresistant A549IR and parental A549 cells. For the p53-deficient (H1299) NSCLC cells, the parental and irradiated populations differed in the expression of 1628 genes and 1616 pathways. The expression of genes associated with radioresistance reflects the complex biological processes involved in clinical cancer cell eradication and might serve as a potential biomarker and therapeutic target for NSCLC treatment.

Abstract 9535: The Effect of Vascular Smooth Muscle Cell miR-33a Expression on apoAI-Mediated Cholesterol Efflux and Macrophage-Like Cell Transdifferentiation

Background: ABCA1 removes cellular cholesterol by participating in apoAI-mediated cholesterol efflux. MiR-33a is an SREBP-2 intronic miRNA that silences ABCA1, which impairs ABCA1-dependent cholesterol efflux. Thus, miR-33a expression may be atherogenic and previous data suggests inhibiting miR-33a in certain cells is atheroprotective. However, current data is scant on whether miR-33a expression in vascular smooth muscle cells (VSMC) is atherogenic. Moreover, cholesterol accumulation in VSMC results in these cells to transdifferentiate into an atherogenic macrophage-like cell (MLC). Therefore, ablating miR-33a expression in VSMC may be atheroprotective via hindering cholesterol accumulation and MLC transdifferentiation in these cells by enhancing apoAI-mediated cholesterol efflux. Methods: An immortalized VSMC line MOVAS cells were used to generate miR-33a knockout (KO) MOVAS cells via using CRISPR/Cas9. We used these cells and parental wild-type (WT) MOVAS cells in our studies to assess whether apoAI-mediated cholesterol efflux is increased in MLC of VSMC origin when miR-33a expression is ablated. We also analyzed whether miR-33a ablation rapidly restores VSMC phenotype in MLC of VSMC origin when cholesterol-loaded cells were exposed to apoAI. Results: We used PCR to confirm that miR-33a was deleted in miR-33a KO MOVAS cells. We also showed that miR-33a-5p and miR-33a-3p expression was absent in miR-33a KO MOVAS cells via RT-PCR. SREBP-2 functionality remained intact in miR-33a KO MOVAS cells, as determined by upregulation of SREBP-2 target genes in serum-starved conditions. When we cholesterol-loaded miR-33a KO MOVAS cells to induce MLC transdifferentiation, we observed increased apoAI-mediated cholesterol efflux when compared to WT MOVAS cells exposed to the same conditions. Based on RT-qPCR analysis, we also observed a rapid restoration in VSMC phenotype when cholesterol-loaded miR-33a KO MOVAS cells were incubated with apoAI when compared to WT MOVAS cells treated with the same conditions. Conclusions: MiR-33a expression in VSMC appears to promote MLC transdifferentiation by impairing apoAI-mediated cholesterol efflux. Future studies are underway to determine if these effects are mediated by miR-33a-5p and/or miR-33a-3p.

Characterization of resistant MCF-7 breast cancer cells developed by repeated cycles of photodynamic therapy.

Breast cancer mainly affects women and causes a severe global threat to health. It is often managed and treated with surgery, chemotherapy, immunotherapy, and radiation therapy. Generally, chemotherapy as a treatment option is often opposed by responsive tumor relapse and development of resistance, a significant setback of current treatment. Photodynamic therapy (PDT) offers a promising modality that can treat cancer by combining a photosensitizer and laser irradiation in the presence of oxygen. However, one problem of PDT in treating breast cancer is the apparition of the resistant cell population. Thus, we aimed for stepwise selection and characterization of MCF-7 cells resistant to PDT with a sulfonated zinc phthalocyanine (ZnPcS4) photosensitizer. The wild-type MCF-7 was exposed to successive cycles of ZnPcS4-PDT, and 10resistant populations were finally obtained. In wild-type and parental cells, we analyzed the cell morphology (light microscopy), cell cycle (BrdU staining), cell viability (MTT assay), antioxidant activity (superoxide dismutase measurement), and immunofluorescence expression of resistant p-glycoprotein (P-gp). The results indicate that resistant cells showed a mesenchymal cell phenotype, few differences in antioxidant activity, an increased DNA synthesis, and more expression of P-gp than the wild-type parental cells. These distinctive features of resistant cells can provide insight into the emergence of MCF-7 cell resistance to PDT, which was necessary to design the best therapeutic procedure for improved efficacy.

Abstract 6346: Identification of ERK inhibitor-based combinations targeting LKB1-mutated NSCLC

Abstract Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. It is a very heterogeneous disease where some frequent mutations remain untargetable. Among them there are those affecting STK11/LKB1, the third most commonly mutated gene in NSCLC adenocarcinomas. Patients harboring LKB1-mutated tumors often have a poor prognosis due to the aggressiveness of this type of cancer and the lack of specific and efficacious therapies. Previous studies in our laboratory demonstrated a peculiar in vitro and in vivo activity of ERK inhibitors (ERKi) on LKB1-mutated NSCLC preclinical models. However, due to the huge heterogeneity of this type of tumor, it is likely that ERKi alone will have limited clinical application. In this scenario, the aim of this project is to find ERKi synthetic lethal partners in order to enlarge NSCLC patient population amenable to this therapy. Methods: We performed high-throughput screenings of a FDA-approved drug library on two different cell line systems (LU99 and H358) each composed by the LKB1 wild-type (wt) parental cell line and the CRISPR-CAS9-derived LKB1 deleted clone. In both the screenings, we treated the cell lines with the FDA-approved library alone or in combination with a subtoxic concentration of the ERKi. Results: According to our aim, for each screening, we analyzed the results selecting as hits those combinations preferentially active on the LKB1-deleted clones compared to their LKB1-wt cell lines. We verified that the FDA-approved compounds were subtoxic when used as single treatment in both the parental cell lines and their clones. We highlighted 28 and 42 hit combinations for LU99 and H358 isogenic systems, respectively. Some hits were common between the two screenings and the most represented FDA-approved drug’s classes of the hit combinations were PI3K/Akt/mTOR inhibitors, tyrosine kinase receptor inhibitors, MAPK inhibitors and compounds involved in anti-inflammatory pathways neuronal signaling, metabolism and DNA damage. Among the hits, we have cross-validated 4 ERKi-based combinations (a tyrosin kinase inhibitor, a MAPK inhibitor, a serine-threonine selective protein inhibitor and a farnesyl transferase inhibitor) in four different isogenic systems and we are now performing further validation on a panel of NSCLC cell lines with different LKB1-status as well as on NSCLC organoids. Conclusions: From the FDA-approved drug library screenings, different ERKi-based combinations showed promising results worthy to be deeply studied. Further analyses are ongoing to verify their efficacy on more complex models as NSCLC 3D organoids and to clarify the mechanism of action at the basis of the hit combinations. Positive results could give the chance to develop effective and specific therapies for patient with LKB1-mutated NSCLCs. Citation Format: Marika Colombo, Matteo Demetrio Tripodi, Gabriel Caso, Elisa Perin, Mirko Marabese, Massimo Broggini, Elisa Caiola. Identification of ERK inhibitor-based combinations targeting LKB1-mutated NSCLC [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 6346.

MicroRNA-29a Manifests Multifaceted Features to Intensify Radiosensitivity, Escalate Apoptosis, and Revoke Cell Migration for Palliating Radioresistance-Enhanced Cervical Cancer Progression.

Radioresistance remains a major clinical challenge in cervical cancer therapy and results in tumor relapse and metastasis. Nevertheless, the detailed mechanisms are still largely enigmatic. This study was conducted to elucidate the prospective impacts of microRNA-29a (miR-29a) on the modulation of radioresistance-associated cervical cancer progression. Herein, we established two pairs of parental wild-type (WT) and radioresistant (RR) cervical cancer cells (CaSki and C33A), and we found that constant suppressed miR-29a, but not miR-29b/c, was exhibited in RR-clones that underwent a dose of 6-Gy radiation treatment. Remarkably, radioresistant clones displayed low radiosensitivity, and the reduced apoptosis rate resulted in augmented surviving fractions, measured by the clonogenic survival curve assay and the Annexin V/Propidium Iodide apoptosis assay, respectively. Overexpression of miR-29a effectively intensified the radiosensitivity and triggered the cell apoptosis in RR-clones. In contrast, suppressed miR-29a modestly abridged the radiosensitivity and abolished the cell apoptosis in WT-clones. Hence, ectopically introduced miR-29a into RR-clones notably attenuated the wound-healing rate and cell migration, whereas reduced miR-29a aggravated cell mobilities of WT-clones estimated via the in vitro wound-healing assay and time-lapse recording assay. Notably, we further established the in vivo short-term lung locomotion metastasis model in BALB/c nude mice, and we found that increased lung localization was shown after tail-vein injection of RR-CaSki cells compared to those of WT-CaSki cells. Amplified miR-29a significantly eliminated the radioresistance-enhanced lung locomotion. Our data provide evidence suggesting that miR-29a is a promising microRNA signature in radioresistance of cervical cancer cells and displays multifaceted innovative roles involved in anti-radioresistance, escalated apoptosis, and anti-cell migration/metastasis. Amalgamation of a nucleoid-based strategy (miR-29a) together with conventional radiotherapy may be an innovative and eminent strategy to intensify the radiosensitivity and further protect against the subsequent radioresistance and the potential metastasis in cervical cancer treatment.

Characterization of SN38-resistant T47D breast cancer cell sublines overexpressing BCRP, MRP1, MRP2, MRP3, and MRP4

BackgroundAlthough several novel resistant breast cancer cell lines have been established, only a few resistant breast cancer cell lines overexpress breast cancer resistance proteins (BCRP). The aim of this study was to establish new resistant breast cancer cell lines overexpressing BCRP using SN38 (7-ethyl-10-hydroxycamptothecin), an active metabolite of irinotecan and was to discover genes and mechanisms associated with multidrug resistance.MethodsSN38-resistant T47D breast cancer cell sublines were selected from the wild-type T47D cells by gradually increasing SN38 concentration. The sensitivity of the cells to anti-cancer drugs was assessed by 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Expression profiles of the resistance-related transporters were examined using RT-qPCR, and western blot analysis. Intracellular fluorescent dye accumulation in the resistant cells was determined using flow cytometry.ResultsThe SN38-resistant T47D breast cancer cell sublines T47D/SN120 and T47D/SN150 were established after long-term exposure (more than 16 months) of wild-type T47D cells to 120 nM and 150 nM SN38, respectively. T47D/SN120 and T47D/SN150 cells were more resistant to SN38 (14.5 and 59.1 times, respectively), irinotecan (1.5 and 3.7 times, respectively), and topotecan (4.9 and 12 times, respectively), than the wild-type parental cells. Both T47D/SN120 and T47D/SN150 sublines were cross-resistant to various anti-cancer drugs. These resistant sublines overexpressed mRNAs of MRP1, MRP2, MRP3, MRP4, and BCRP. The DNA methylase inhibitor 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor trichostatin A increased the expression levels of BCRP, MRP1, MRP2, MRP3, and MRP4 transcripts in T47D/WT cells. Fluorescent dye accumulation was found to be lower in T47D/SN120 and T47D/SN150 cells, compared to that in T47D/WT cells. However, treatment with known chemosensitizers increased the intracellular fluorescent dye accumulation and sensitivity of anti-tumor agents.ConclusionT47D/SN120 and T47D/SN150 cells overexpressed MRP1, MRP2, MRP3, MRP4, and BCRP, which might be due to the suppression of epigenetic gene silencing via DNA hypermethylation and histone deacetylation. Although these resistant cells present a higher resistance to various anti-cancer drugs than their parental wild-type cells, multidrug resistance was overcome by treatment with chemosensitizers. These SN38 resistant T47D breast cancer cell sublines expressing resistance proteins can be useful for the development of new chemosensitizers.

Quantitative glycoproteomics analysis identifies novel FUT8 targets and signaling networks critical for breast cancer cell invasiveness

BackgroundWe recently showed that fucosyltransferase 8 (FUT8)-mediated core fucosylation of transforming growth factor-β receptor enhances its signaling and promotes breast cancer invasion and metastasis. However, the complete FUT8 target glycoproteins and their downstream signaling networks critical for breast cancer progression remain largely unknown.MethodWe performed quantitative glycoproteomics with two highly invasive breast cancer cell lines to unravel a comprehensive list of core-fucosylated glycoproteins by comparison to parental wild-type and FUT8-knockout counterpart cells. In addition, ingenuity pathway analysis (IPA) was performed to highlight the most enriched biological functions and signaling pathways mediated by FUT8 targets. Novel FUT8 target glycoproteins with biological interest were functionally studied and validated by using LCA (Lens culinaris agglutinin) blotting and LC–MS/MS (liquid chromatography–tandem mass spectrometry) analysis.ResultsLoss-of-function studies demonstrated that FUT8 knockout suppressed the invasiveness of highly aggressive breast carcinoma cells. Quantitative glycoproteomics identified 140 common target glycoproteins. Ingenuity pathway analysis (IPA) of these target proteins gave a global and novel perspective on signaling networks essential for breast cancer cell migration and invasion. In addition, we showed that core fucosylation of integrin αvβ5 or IL6ST might be crucial for breast cancer cell adhesion to vitronectin or enhanced cellular signaling to interleukin 6 and oncostatin M, two cytokines implicated in the breast cancer epithelial–mesenchymal transition and metastasis.ConclusionsOur report reveals a comprehensive list of core-fucosylated target proteins and provides novel insights into signaling networks crucial for breast cancer progression. These findings will assist in deciphering the complex molecular mechanisms and developing diagnostic or therapeutic approaches targeting these signaling pathways in breast cancer metastasis.

Transcriptomic Analysis Reveals Competitive Growth Advantage of Non-pigmented Serratia marcescens Mutants.

Serratia marcescens is a common bacterium well-known for the red secondary metabolite prodigiosin. However, color mutants have long been described. Non-pigmented strains can be found to exist both naturally and under laboratory conditions. It is unclear why S. marcescens loses prodigiosin synthesis capacity in certain conditions. In the present study, we find that the spontaneous color mutants arise within a few generations (about five passages) and rapidly replace the wild-type parent cells (about 24 passages), which indicates a growth advantage of the former. Although, the loss of prodigiosin synthesis genes (pigA-N) is frequently reported as the major reason for pigment deficiency, it was unexpected that the whole gene cluster is completely preserved in the different color morphotypes. Comparative transcriptomic analysis indicates a dramatic variation at the transcriptional level. Most of the pig genes are significantly downregulated in the color morphotypes which directly lead to prodigiosin dyssynthesis. Besides, the transcriptional changes of several other genes have been noticed, of which transcriptional regulators, membrane proteins, and nearly all type VI secretion system (T6SS) components are generally downregulated, while both amino acid metabolite and transport systems are activated. In addition, we delete the transcription regulator slyA to generate a non-pigmented mutant. The ΔslyA strain loses prodigiosin synthesis capacity, but has a higher cell density, and surprisingly enhances the virulence as an entomopathogen. These data indicate that S. marcescens shuts down several high-cost systems and activates the amino acid degradation and transport pathways at the transcriptional level to obtain extra resources, which provides new insights into the competitive growth advantage of bacterial spontaneous color mutants.

3114 – FROM MECHANISMS OF LEUKAEMOGENIC DYSREGULATION TO NEW CANDIDATE THERAPEUTICS

Rearrangements involving the MLL1 gene (Kmt2a) produce powerful oncogenic proteins occurring predominantly in paediatric and infant acute myeloid and lymphoid leukaemias. Despite having more than 80 fusion partners, MLL-AF9, MLL-AF4 and MLL-ENL make up more than 60% of cases involving MLL1 translocations. Conventional retroviral transduction models commonly use lineage depleted or c-Kit enriched mouse bone marrow HSPCs that display distinct heterogeneity when profiled at the single-cell level which could explain inconsistencies across previously reported models. Conversely, our model implementing a conditionally blocked multipotent haematopoietic progenitor murine cell line (Hoxb8-FL) allows reproducible tracking of early leukaemic transformation and transcriptional changes due to MLL-ENL expression (Basillico., et al 2019). Using our new cellular mouse model of AML, we knocked out 64 genes via CRISPR-Cas9 followed by small-bulk RNA-Seq. The 64 targets include transcription factors (TFs) and key transcriptional regulators implicated in the aetiology of MLL rearranged leukaemia. Thus, we have defined a leukaemic TF network consisting of around 60,000 links across approximately 11,000 genes. Previous work in our laboratory used the same approach to establish the gene regulatory function of 39 TFs in the wild-type parental Hoxb8-FL cells (Kucinski et al. 2020). Our model will allow us to directly compare our network to the wild-type cell of origin to interrogate how the TF network is underpinning leukaemic transcriptional dysregulation. Novel computational methods will allow us to decipher whether the perturbation of specific genes results in the identification of potential candidate therapeutic vulnerabilities. Rearrangements involving the MLL1 gene (Kmt2a) produce powerful oncogenic proteins occurring predominantly in paediatric and infant acute myeloid and lymphoid leukaemias. Despite having more than 80 fusion partners, MLL-AF9, MLL-AF4 and MLL-ENL make up more than 60% of cases involving MLL1 translocations. Conventional retroviral transduction models commonly use lineage depleted or c-Kit enriched mouse bone marrow HSPCs that display distinct heterogeneity when profiled at the single-cell level which could explain inconsistencies across previously reported models. Conversely, our model implementing a conditionally blocked multipotent haematopoietic progenitor murine cell line (Hoxb8-FL) allows reproducible tracking of early leukaemic transformation and transcriptional changes due to MLL-ENL expression (Basillico., et al 2019). Using our new cellular mouse model of AML, we knocked out 64 genes via CRISPR-Cas9 followed by small-bulk RNA-Seq. The 64 targets include transcription factors (TFs) and key transcriptional regulators implicated in the aetiology of MLL rearranged leukaemia. Thus, we have defined a leukaemic TF network consisting of around 60,000 links across approximately 11,000 genes. Previous work in our laboratory used the same approach to establish the gene regulatory function of 39 TFs in the wild-type parental Hoxb8-FL cells (Kucinski et al. 2020). Our model will allow us to directly compare our network to the wild-type cell of origin to interrogate how the TF network is underpinning leukaemic transcriptional dysregulation. Novel computational methods will allow us to decipher whether the perturbation of specific genes results in the identification of potential candidate therapeutic vulnerabilities.

CRISPR/Cas9‐mediated knockout of G proteins and β‐arrestins defines their specific but interdependent roles in dopamine D1 receptor signaling

The dopamine D1 receptor (D1R) is a G protein-coupled receptor which regulates key brain functions including voluntary movement, memory, attention, and reward. Selective agonist activation of the D1R may also provide novel neurotherapeutics for neurodegenerative and neuropsychiatric disorders. The D1R canonically activates the heterotrimeric G proteins Gαs, and possibly Gαolf, which activate adenylyl cyclase (AC) to increase cAMP/PKA signaling. The D1R also engages β-arrestin proteins which may transduce β-arrestin dependent signaling by scaffolding and activating kinases. However, the distinct roles of Gαs, Gαolf versus β-arrestins 1 and 2 for D1R signaling remains largely undefined. Here we utilize CRISPR/Cas9 engineered HEK293 cells in which Gαs, Gαolf, β-arrestin1 and β-arrestin2 are stably knocked out (KO) to delineate D1R pharmacology and signaling mechanisms. Western blots confirmed a complete loss of Gαs, Gαolf, β-arrestin1 or β-arrestin2 proteins in KO cells versus parental wildtype cells. Dose responses with the D1R agonist SKF-81297 or β2AR agonist isoproterenol induced robust cAMP production in wildtype cells that was eliminated in Gαs/olf KO cells. Re-expression of Gαs or Gαolf in KO cells rescued D1R-mediated cAMP signaling similar to wildtype cells. Interestingly, the D1R had significantly higher cAMP signaling when coupling via Gαs as compared to Gαolf, while the β2AR only coupled with Gαs to stimulate cAMP signaling. Western blotting indicated Gαs/olf KO completely blocked agonist induced CREB phosphorylation by both D1R and β2AR agonists, indicating a strict dependence on Gαs/olf and cAMP/PKA signaling to activate CREB. In major contrast, β-arrestin1/2 KO significantly increased the intensity and duration of cAMP signaling and CREB phosphorylation by the β2AR. β-arrestin1/2 KO also entirely blocked agonist-induced D1R internalization and impaired D1R desensitization while Gαs/olf KO had no effect on D1R internalization. Key controls of re-expressing Gαs, Gαolf, β-arrestin1 or β-arrestin2 in KO cells completely restored signaling or internalization similar to wildtype cells, indicating CRISPR/Cas9 targeting did not result in non-specific or compensatory effects in the pathways under study. Notably, β-arrestin1/2 KO also potentiated and prolonged D1R or β2AR-induced ERK1/2 phosphorylation. D1R-induced ERK1/2 phosphorylation in β-arrestin1/2 KO cells was eliminated by the SRC inhibitor SU6656 or the MEK inhibitor U0126, suggesting that D1R signaling via a Gαs/SRC/MEK pathway is essential for ERK1/2 activation. Taken together, our studies indicate that Gαs is the dominant transducer for D1R cAMP, CREB and ERK1/2 signaling and that β-arrestins provide distinctly interdependent roles with G proteins in modulating the duration and intensity of D1R and β2AR signaling cascades. Using CRISPR/Cas9 KO model cells, future studies will examine the distinct contributions of β-arrestins 1 and 2 and Gαs/olf for D1R pharmacology, which will be useful in the development of novel D1R biased agonists.

Inositol phosphates promote HIV-1 assembly and maturation to facilitate viral spread in human CD4+ T cells.

Gag polymerization with viral RNA at the plasma membrane initiates HIV-1 assembly. Assembly processes are inefficient in vitro but are stimulated by inositol (1,3,4,5,6) pentakisphosphate (IP5) and inositol hexakisphosphate (IP6) metabolites. Previous studies have shown that depletion of these inositol phosphate species from HEK293T cells reduced HIV-1 particle production but did not alter the infectivity of the resulting progeny virions. Moreover, HIV-1 substitutions bearing Gag/CA mutations ablating IP6 binding are noninfectious with destabilized viral cores. In this study, we analyzed the effects of cellular depletion of IP5 and IP6 on HIV-1 replication in T cells in which we disrupted the genes encoding the kinases required for IP6 generation, IP5 2-kinase (IPPK) and Inositol Polyphosphate Multikinase (IPMK). Knockout (KO) of IPPK from CEM and MT-4 cells depleted cellular IP6 in both T cell lines, and IPMK disruption reduced the levels of both IP5 and IP6. In the KO lines, HIV-1 spread was delayed relative to parental wild-type (WT) cells and was rescued by complementation. Virus release was decreased in all IPPK or IPMK KO lines relative to WT cells. Infected IPMK KO cells exhibited elevated levels of intracellular Gag protein, indicative of impaired particle assembly. IPMK KO compromised virus production to a greater extent than IPPK KO suggesting that IP5 promotes HIV-1 particle assembly in IPPK KO cells. HIV-1 particles released from infected IPPK or IPMK KO cells were less infectious than those from WT cells. These viruses exhibited partially cleaved Gag proteins, decreased virion-associated p24, and higher frequencies of aberrant particles, indicative of a maturation defect. Our data demonstrate that IP6 enhances the quantity and quality of virions produced from T cells, thereby preventing defects in HIV-1 replication.

MicroRNA-128 Confers Anti-Endothelial Adhesion and Anti-Migration Properties to Counteract Highly Metastatic Cervical Cancer Cells' Migration in a Parallel-Plate Flow Chamber.

Despite the distant metastasis of cervical cancer cells being a prominent cause of mortality, neither the metastasis capacity nor the in vitro conditions mimicking adhesion of cervical cancer cells to endothelial cells have been fully elucidated. Circulating metastatic cancer cells undergo transendothelial migration and invade normal organs in distant metastasis; however, the putative molecular mechanism remains largely uncertain. In this study, we describe the use of an in vitro parallel-plate flow chamber to simulate the dynamic circulation stress on cervical cancer cells and elucidate their vascular adhesion and metastasis. We isolate the viable and shear stress-resistant (SSR) cervical cancer cells for mechanistic studies. Remarkably, the identified SSR-HeLa and SSR-CaSki exhibited high in vitro adhesive and metastatic activities. Hence, a consistently suppressed miR-128 level was revealed in SSR cell clones compared to those of parental wild-type (WT) cells. Overexpressed miR-128 attenuated SSR-HeLa cells’ adherence to human umbilical cord vein endothelial cells (HUVECs); in contrast, suppressed miR-128 efficiently augmented the static adhesion capacity in WT-HeLa and WT-CaSki cells. Hence, amplified miR-128 modestly abolished in vitro SSR-augmented HeLa and CaSki cell movement, whereas reduced miR-128 aggravated the migration speed in a time-lapse recording assay in WT groups. Consistently, the force expression of miR-128 alleviated the SSR-enhanced HeLa and CaSki cell mobility in a wound healing assay. Notably, miR-128 mediated SSR-enhanced HeLa and CaSki cells’ adhesion and metastasis through suppressed ITGA5, ITGB5, sLex, CEACAM-6, MMP9, and MMP23 transcript levels. Our data provide evidence suggesting that miR-128 is a promising microRNA that prevented endothelial cells’ adhesion and transendothelial migration to contribute to the SSR-enhanced adhesion and metastasis progression under a parallel-plate flow chamber system. This indicates that the nucleoid-based miR-128 strategy may be an attractive therapeutic strategy to eliminate tumor cells resistant to circulation shear flow, prevent vascular adhesion, and preclude subsequent transendothelial metastasis.