Resistant Cell Clones Research Articles

Medullary Thyroid Cancer: Molecular Drivers and Immune Cellular Milieu of the Tumour Microenvironment-Implications for Systemic Treatment.

In this review, we explore the underlying molecular biology of medullary thyroid carcinoma (MTC) and its interplay with the host immune system. MTC is consistently driven by a small number of specific pathogenic variants, beyond which few additional genetic events are required for tumorigenesis. This explains the exceedingly low tumour mutational burden seen in most MTC, in contrast to other cancers. However, because of the low tumour mutational burden (TMB), there is a correspondingly low level of tumour-associated neoantigens that are presented to the host immune system. This reduces tumour visibility and vigour of the anti-tumour immune response and suggests the efficacy of immunotherapy in MTC is likely to be poor, acknowledging this inference is largely based on the extrapolation of data from other tumour types. The dominance of specific RET (REarranged during Transfection) pathogenic variants in MTC tumorigenesis rationalizes the observed efficacy of the targeted RET-specific tyrosine kinase inhibitors (TKIs) in comparison to multi-kinase inhibitors (MKIs). Therapeutic durability of pathway inhibitors is an ongoing research focus. It may be limited by the selection pressure TKI treatment creates, promoting survival of resistant tumour cell clones that can escape pathway inhibition through binding-site mutations, activation of alternate pathways, and modulation of the cellular and cytokine milieu of the tumour microenvironment (TME).

Melanoma Clonal Heterogeneity Leads to Secondary Resistance after Adoptive Cell Therapy with Tumor-Infiltrating Lymphocytes.

Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TIL) is effective in patients with melanoma, although long-term responses seem restricted in patients who have complete remissions. Many patients develop secondary resistance to TIL-ACT but the involved mechanisms are unclear. In this study, we describe a case of secondary resistance to TIL-ACT possibly due to intratumoral heterogeneity and selection of a resistant tumor cell clone by the transferred T cells. To the best our knowledge, this is the first case of clonal selection of a pre-existing nondominant tumor cell clone; this report demonstrates the mechanism involved in secondary resistance to TIL-ACT that can potentially change current clinical practice because it advocates for T-cell collection from multiple tumor sites and analysis of tumor heterogeneity before treatment with TIL-ACT.

Abstract 7508: Distribution prophecy of circulating tumor cell clusters in CTC populace patients of epithelial cancers

Abstract Background: Role of CTCs in metastatic cancers to predict the % prediction of overall survival has been well-established. The effectiveness of three- or six-months adjuvant therapy in colorectal cancer estimation showed association of CTCs with the emergence of resistant cell clones. The presence of CTCs with clusters indicates an aggressiveness of the epithelial cancers. However, the presence of CTC clusters has not been evaluated in large populations. Here, we demonstrate the distribution prophecy of CTCs and CTC clusters in epithelial cancer patients. Methods: Retrospectively, 3458 patients' blood sample was analyzed for the presence and distribution of CTCs and CTC clusters using DCGI approved OncoDiscover platform having immunomagnetic multicomponent system mediated by anti EpCAM antibody. 1.5 mL of peripheral blood was analyzed to capture cells and clusters from Head & Neck, breast, and lung cancer patients. Earlier the sensitivity, specificity and accuracy of OncoDiscover assay was presented. CTCs and clusters were identified with CK 18 +ve, DAPI + ve and CD45 -ve staining using automated motorized fluorescence microscopy. CTCs clusters were defined by the presence of two or more CTCs bound together. Results: Out of 3458 epithelial cancer patients, 65.52 % patients showed the distribution of CTCs ~ 2262 (CTC numbers varied from 1-9/1.5 mL), while 7.54% patients showed CTC clusters (261 clusters). Total number of captured CTCs and clusters were 19345 (mean CTCs and cluster distribution was 5.59), while CTC were predominantly in 19037 numbers (98.41%) and clusters were 308 (1.59 %). Highest number of CTS were observed in HNC (52.98%) and breast cancers (22.75%), followed by lung cancer (5.65%). While, prophecy clusters were observed in breast cancers (26.95%), followed by lung (16.23%). Conclusions: The frequency and distribution of CTCs and CTC clusters was evaluated in epithelial cancers. Patients with CTC alone and patients having CTCs as well CTC clusters represent more aggressive disease and the progression of disease. The effectiveness of adjuvant therapy in epithelial cancers can be estimated with CTC and cluster for association of treatment outmoded with an emergence of resistant cell clones. Citation Format: Jayant Khandare, Gourishankar Aland, Ganesh Khutale, Saloni Andhari, Prathamesh Patil, Meghna Garbhe, Manoj Dongare, Tanvi Deshpande, Vikas Jadhav, Nirmal Raut, Gowhar Shafi, Atul Bharde, Aravindan Vasudevan, Sreeja Jayant. Distribution prophecy of circulating tumor cell clusters in CTC populace patients of epithelial 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 7508.

Understanding the Role of Cancer Stem Cells in Treatment Resistance and Poor Outcomes of Rectal Cancer in Indian Population

Background Rectal cancer is one of the common gastrointestinal cancers and a leading cause for cancer deaths in India especially in the younger population. Cancer stem cells as part of intratumoral heterogeneity are attributed to therapy failure due to stemness related resistance mechanisms. Better understanding of resistant stem cell clones of rectal cancer will increase the success rate of cancer treatment. Methods After Institutional ethical committee approval the primary cell cultures from two tumour specimens of rectal cancer patients planned for chemoradiation and at the time of surgery were prepared. From single cell suspension cultures total stem cell population was estimated based upon Hoechst 33342 dye staining technique and its correlation with patientsrsquo overall survival was evaluated.Results During the study period out of 91 newly diagnosed rectal cancer patients 21 rectal cancer tissue samples were collected before chemoradiation and at the time of surgery from 12 patients consisting of ten males and two females in the age range of 29 to 67 years. Single cell suspension culture from tumour tissue samples collected from eight patients prior to their chemoradiation were prepared with maximum duration of viability of 10 days. Range of percentage of Hoechst low stem cells to Hoechst bright tumours cells was from 0.2 to 0.9 in eight SCSC with significant correlation with overall survival.Conclusion Cancer stem cell populations in rectal cancer patients can be attributed to poor outcomes owing to the resistance mechanisms to chemoradiation termed as stemness. Better understanding of stemness and targeting of pathways by novel therapies can improve current poor outcome.

Evaluating immune response in vitro in a relevant microenvironment: a high-throughput microfluidic model for clinical screening.

Functional screening of new pharmaceutical compounds requires clinically relevant models to monitor essential cellular and immune responses during cancer progression, with or without treatment. Beyond survival, the emergence of resistant tumor cell clones should also be considered, including specific properties related to plasticity, such as invasiveness, stemness, escape from programmed cell death, and immune response. Numerous pathways are involved in these processes. Defining the relevant ones in the context of a specific tumor type will be key to designing an appropriate combination of inhibitors. However, the diversity and potential redundancy of these pathways remain a challenge for therapy. A new microfluidic device developed by Okomera was dedicated to the screening of drug treatment for breast cancer. This microchip includes 150 droplet-trapping microwells, offering multi-chip settings and multiple treatment choices. After validating the system with established cell lines and a panel of drugs used clinically at Gustave Roussy, preclinical experiments were initiated including patient-derived xenograft (PDX) and primary tumor cells-derived tumoroids with the collaboration of Gustave Roussy clinicians. Tumor-isolated lymphocytes were also added to the tumoroids, using secondary droplets in proof-of-concept experiments. These results show the relevance of the methodology for screening large numbers of drugs, a wide range of doses, and multiple drug combinations. This methodology will be used for two purposes: 1) new drug screening from the compound library, using the high throughput potential of the chip; and 2) pre-clinical assay for a two-weeks response for personalized medicine, allowing evaluation of drug combinations to flag an optimized treatment with potential clinical application.

Different costs of therapeutic resistance in cancer: Short- and long-term impact of population heterogeneity

Therapeutic resistance continues to undercut long-term success of many promising cancer treatments. At times, development of therapeutic resistance can come at a fitness cost for the cancer cell population, which could potentially be leveraged to the patient’s advantage. A mathematical formulation of such a situation was proposed by Pressley et al. (2020), who discussed two scenarios, namely, when developing therapeutic resistance can come at a cost to proliferative capacity (such as when a drug targets a growth receptor), or to the total tumor carrying capacity (such as when a drug targets neovascularization). Here we expand the analysis of the two models and evaluate both short- and long-term dynamics of a population heterogeneous with respect to resistance. We analyze four initial distributions with respect to resistance at the time of treatment initiation: uniform, bell-shaped, exponential, and U-shaped. We show that final population composition is invariant to the initial distribution, with a single clone eventually dominating within the population; the value of the resistance parameter of the final clone depends on other system parameters but not on the initial distribution. Transitional behaviors, however, which may have more significant implications for immediate treatment decisions, depend critically on the initial distribution. Furthermore, we show that depending on the mechanism for the cost of resistance (i.e., proliferation vs carrying capacity), increase in natural cell death rate has opposite effects, with higher natural death rate selecting for less resistant cell clones in the long term for proliferation-dependent model, and selecting for more resistant cell clones for carrying capacity-dependent model, a prediction that may have implications for combination therapy with cytotoxic agents. We conclude with a discussion of strengths and limitations of using modeling for understanding treatment trajectory, as well as the promise of model-informed evolutionary steering for improved long-term therapeutic outcomes.

Molecular mechanisms of drug resistance of glial tumor of brain. Part 2: Proliferation, angiogenesis, metastasis and recurrency

The main reason for the low efficiency of glioblastoma therapy is its resistance to therapeutic procedures. The development of multidrug resistance occurs as a result of the selection of tumor clones during therapy. The resistant cell clones to radiotherapy and chemotherapy can proliferate, leading to tumor growth, in which its own vascular network is formed (angiogenesis), which promotes cell migration, invasion and the appearance of metastases and recurrent glioblastoma. The review examines the relationship at the molecular level of multidrug resistance with proliferation, angiogenesis, migration, metastasis, and the formation of glioblastoma relapses, with an emphasis on identifying new targets among proteins, microRNAs, signal transduction kinases, transcription factors, tumor-supressor genes and oncogenes.

Evaluation of circulating tumor cells in stage III colorectal cancer patients under three or six months of adjuvant therapy.

e15601 Background: The duration of adjuvant oxaliplatin-combination treatment in stage III colorectal cancer (CRC) patients is still debatable. Circulating Tumor Cells (CTCs) have been proposed as a potential markers for detection of micrometastatic disease. The aim was to compare the elimination rate of CTCs in patients treated with three- and six-months treatment with FOLFOX or CAPOX, in stage III CRC patients. Methods: Peripheral blood samples were collected from 132 patients at baseline, after three (for all patients) and after six months (for those under six months treatment). Surgical specimens were collected from all patients for the evaluation of their genetic profiling using Sanger sequencing (Ras/Raf) and fragment analysis (microsatellite instability status). The detection and number of circulating tumor cells (CTCs) were evaluated using double immunofluorescence and a real time-polymerase chain reaction assay, at all three time points. Results: Median age of the patients was 62 years, 59.8% were males, 92.7% had a colon/sigmoid tumor location, 24.4% had tumor on the right colon and 99.2% had a good performance status (PS). Of all patients, 44.3% and 55.7% were treated with FOLFOX and CAPOX, respectively; patients received treatment for three and six months, were equally distributed. KRAS, NRAS and BRAFmutations were detected in 41.5%, 2.1% and 12.8%, respectively; whereas MSI-High status was detected in 9.8% of the patients. CTCs were detected in 43.2%, 42.4% and 54.2% of the patients, at baseline, after three and six months of treatment, respectively. Both positivity rate ( p< 0.001) and absolute number of CTCs ( p= 0.022) were decreased between baseline and three months of treatment. Moreover, a significant increase was observed both on the positivity rate ( p= 0.053) and absolute number of CTCs ( p< 0.001) between three and six months of treatment. Following Cox regression analysis, BRAF status and the detection of CTCs are significantly correlated with decreased time to progression. Conclusions: To conclude, the current study indicates that 3 months of adjuvant treatment is at least as efficacious as 6 months in eliminating CTCs. However, the increase of both the rate and number of positive CTCs after three months of treatment might highlight the emergence of resistant cancer cell clones.

O-7 Evidence of therapeutic effectiveness of third-line cetuximab rechallenge in appropriately selected patients: Findings from long-term follow-up of CRICKET and CAVE trials

Rechallenge strategy is based on the concept that a subset of patients with RAS wild type (WT) metastatic colorectal cancer (mCRC) could still benefit of epidermal growth factor receptor (EGFR) blockade, even after progression to an anti-EGFR based chemotherapy. In fact, after an anti-EGFR free window, that reduces the number of resistant cancer cell clones, re-treatment with the anti-EGFR monoclonal antibodies cetuximab or panitumumab may have antitumor activity. Here we performed a pooled and comparative analysis of two phase II prospective trials in rechallenge setting.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

The Tongue Squamous Carcinoma Cell Line Cal27 Primarily Employs Integrin α6β4-Containing Type II Hemidesmosomes for Adhesion Which Contribute to Anticancer Drug Sensitivity.

Integrins are heterodimeric cell surface glycoproteins used by cells to bind to the extracellular matrix (ECM) and regulate tumor cell proliferation, migration and survival. A causative relationship between integrin expression and resistance to anticancer drugs has been demonstrated in different tumors, including head and neck squamous cell carcinoma. Using a Cal27 tongue squamous cell carcinoma model, we have previously demonstrated that de novo expression of integrin αVβ3 confers resistance to several anticancer drugs (cisplatin, mitomycin C and doxorubicin) through a mechanism involving downregulation of active Src, increased cell migration and invasion. In the integrin αVβ3 expressing Cal27-derived cell clone 2B1, αVβ5 expression was also increased, but unrelated to drug resistance. To identify the integrin adhesion complex (IAC) components that contribute to the changes in Cal27 and 2B1 cell adhesion and anticancer drug resistance, we isolated IACs from both cell lines. Mass spectrometry (MS)-based proteomics analysis indicated that both cell lines preferentially, but not exclusively, use integrin α6β4, which is classically found in hemidesmosomes. The anticancer drug resistant cell clone 2B1 demonstrated an increased level of α6β4 accompanied with increased deposition of a laminin-332-containing ECM. Immunofluorescence and electron microscopy demonstrated the formation of type II hemidesmosomes by both cell types. Furthermore, suppression of α6β4 expression in both lines conferred resistance to anticancer drugs through a mechanism independent of αVβ3, which implies that the cell clone 2B1 would have been even more resistant had the upregulation of α6β4 not occurred. Taken together, our results identify a key role for α6β4-containing type II hemidesmosomes in regulating anticancer drug sensitivity.

Acquired Resistance to Immune Checkpoint Blockades: The Underlying Mechanisms and Potential Strategies.

The immune checkpoint blockade therapy has completely transformed cancer treatment modalities because of its unprecedented and durable clinical responses in various cancers. With the increasing use of immune checkpoint blockades in clinical practice, a large number of patients develop acquired resistance. However, the knowledge about acquired resistance to immune checkpoint blockades is limited and poorly summarized. In this review, we clarify the principal elements of acquired resistance to immune checkpoint blockades. The definition of acquired resistance is heterogeneous among groups or societies, but the expert consensus of The Society for Immunotherapy of Cancer can be referred. Oligo-progression is the main pattern of acquired resistance. Acquired resistance can be derived from the selection of resistant cancer cell clones that exist in the tumor mass before therapeutic intervention or gradual acquisition in the sensitive cancer cells. Specifically, tumor intrinsic mechanisms include neoantigen depletion, defects in antigen presentation machinery, aberrations of interferon signaling, tumor-induced exclusion/immunosuppression, and tumor cell plasticity. Tumor extrinsic mechanisms include upregulation of other immune checkpoints. Presently, a set of treatment modalities is applied to patients with similar clinical characteristics or resistance mechanisms for overcoming acquired resistance, and hence, further research is required.

Poly(ADP-Ribose) Polymerase Inhibitors for Arsenic Trioxide-Resistant Acute Promyelocytic Leukemia: Synergistic In Vitro Antitumor Effects with Hypomethylating Agents or High-Dose Vitamin C.

Arsenic trioxide (ATO) is an anticancer agent used for the treatment ofacute promyelocytic leukemia (APL). However, 5%-10% of patients fail to respond or experience disease relapse. Based on poly(ADP-ribose) polymerase (PARP) 1 involvement in the processing of DNA demethylation, here we have tested the in vitro susceptibility of ATO-resistant clones (derived from the human APL cell line NB4) to PARP inhibitors (PARPi) in combination with hypomethylating agents (azacitidine and decitabine) or high-dose vitamin C (ascorbate), which induces 5-hydroxymethylcytosine (5hmC)-mediated DNA demethylation. ATO-sensitive and -resistant APL cell clones were generated and initially analyzed for their susceptibility to five clinically used PARPi (olaparib, niraparib, rucaparib, veliparib, and talazoparib). The obtained PARPi IC50 values were far below (olaparib and niraparib), within the range (talazoparib), or above (rucaparib and veliparib) the C max reported in patients, likely as a result of differences in the mechanisms of their cytotoxic activity. ATO-resistant APL cells were also susceptible to clinically relevant concentrations of azacitidine and decitabine and to high-dose ascorbate. Interestingly, the combination of these agents with olaparib, niraparib, or talazoparib resulted in synergistic antitumor activity. In combination with ascorbate, PARPi increased the ascorbate-mediated induction of 5hmC, which likely resulted in stalled DNA repair and cytotoxicity. Talazoparib was the most effective PARPi in synergizing with ascorbate, in accordance with its marked ability to trap PARP1 at damaged DNA. These findings suggest that ATO and PARPi have nonoverlapping resistance mechanisms and support further investigation on PARPi combination with hypomethylating agents or high-dose ascorbate for relapsed/ATO-refractory APL, especially in frail patients. SIGNIFICANCE STATEMENT: This study found that poly(ADP-ribose) inhibitors (PARPi) show activity as single agents against human acute promyelocytic leukemia cells resistant to arsenic trioxide at clinically relevant concentrations. Furthermore, PARPi enhance the in vitro efficacy of azacitidine, decitabine, and high-dose vitamin C, all agents that alter DNA methylation. In combination with vitamin C, PARPi increase the levels of 5-hydroxymethylcytosine, likely as a result of altered processing of the oxidized intermediates associated with DNA demethylation.

Combining radiotherapy with targeted therapies in non-small cell lung cancer: focus on anti-EGFR, anti-ALK and anti-angiogenic agents.

The combination of radiotherapy (RT) with targeted agents in non-small cell lung cancer (NSCLC) has been expected to improve the therapeutic ratio and tumor control. The EGFR blockade enhances the antitumor effect of RT. The ALK inhibition elicits anti-proliferative, pro-apoptotic and antiangiogenic effects in ALK-positive NSCLC cell lines, enhanced by the exposure to RT. The antiangiogenic agents normalize pathological tumor vessels, thus decrease tumor cell hypoxia and improve radiosensitivity. To date, however, none of the targeted agents combined with RT has shown proven clinical benefit over standard chemoradiation (CRT) in locally advanced NSCLC. The risk of potential excessive toxicity related to the therapeutic combination of RT and targeted agents cannot be ignored. Well-designed clinical trials may allow development of more effective combination strategies. Another potential application of combined RT and targeted therapies in oncogene-driven NSCLC is metastatic oligoprogressive or oligopersistent disease. The use of RT in oligoprogressive oncogene-driven NSCLC, while continuing first line targeted therapy, can potentially eradicate resistant cell clones and provide survival benefit. Likewise, the consolidation of oligopersistent foci (molecularly resistant to first line targeted therapy) may potentially interfere with the natural course of the disease by avoiding or delaying progression. We discuss here the molecular and radiobiological mechanisms of combining RT and targeted agents, and summarize current clinical experience.

STEM-07. IDENTIFICATION OF A NOVEL CSC SIGNALING AXIS CONTAINING JAM-A/SERPINB3/TGF-BETA

Abstract Despite therapeutic interventions including surgery, radiation, and chemotherapy, multiple clones of treatment resistant cells repopulate the tumor, resulting in recurrence and a high rate of mortality in glioblastoma. Effective targeting of these cancer stem cells (CSCs) has been limited by our incomplete understanding of the intracellular signaling mechanisms maintaining the CSC state. Our laboratory previously identified junctional adhesion molecule-A (JAM-A) on CSCs and, through functional studies, demonstrated that JAM-A is both necessary and sufficient for self-renewal and tumor growth. We determined that JAM-A signals via Akt in GBM CSCs to sustain pluripotency transcription factor activity; however, the intermediate signaling network is yet to be fully elucidated. Therefore, our goal was to delineate the pathway downstream of JAM-A. To achieve this, we immunoprecipitated JAM-A from GBM CSCs and performed mass spectrometry leading to the identification of the serine/cysteine protease inhibitor SerpinB3. While SerpinB3 has a functional role in lung adenocarcinoma, breast, pancreas, as well as in hepatocellular carcinoma, there is limited information in GBM or CSCs, and its relationship to JAM-A is yet to be elucidated. Using in vitro CSC functional assays, we observed that SerpinB3 is necessary for the maintenance of CSCs. Additionally, in an intracranial implantation model of GBM the knockdown of SerpinB3 extended the survival of mice. Knockdown of SerpinB3 in multiple GBM CSCs models also led to decreased expression of TGF-β, a known regulator of the CSC state. We also observed that SerpinB3 stabilizes JAM-A expression and further studies are investigating the mechanism of this stabilization, identifying the specific interaction sites of JAM-A and Serpin B3, and determining how TGF-β alters the association through positive and negative feedback mechanisms. Taken together, these data highlight the role of SerpinB3 in GBM CSCs and provides a novel target to inhibit the JAM-A-mediated CSC maintenance as a next-generation GBM therapy.

Abstract PO-132: CaTCH - A barcode-guided CRISPRa-inducible reporter to isolate clones from heterogeneous populations

Abstract The emergence of resistant cell clones to targeted therapies poses a significant issue in the treatment of metastatic melanoma. While these founding clones are often extremely rare in a starting population, their isolation and characterization holds unique potential for understanding disease processes, uncovering novel biomarkers and developing therapeutic concepts. The functional characterization of such founder clones and comprehensive comparisons to their post-selection counterparts requires live cells. To achieve this, we developed a novel lineage tracing tool termed CaTCH (CRISPRa tracing of clones in heterogeneous cell populations). CaTCH combines precise mapping of the lineage history of millions of cells with the ability to isolate any given clone alive from a complex population based on genetic barcodes. CaTCH thereby enables the retrospective isolation and analysis of founding clones from heterogeneous cell populations prior to evolutionary selection. In first applications, we use CaTCH to provide insights into the development of resistance to targeted cancer therapies. We demonstrate that CaTCH can be used to trace and isolate a single pre-existing therapy-resistant clone from a complex cancer cell population in vitro. Furthermore, we validate the utility of CaTCH for applications in vivo by investigating the origins of resistance to clinically relevant RAF/MEK inhibition in an immunocompetent melanoma mouse model. Here we find that most clones have the capacity to acquire resistance to combined RAF/MEK inhibitor therapy, indicating that resistance to this clinically relevant regimen is a universally achievable state in this model. We envision that CaTCH will address fundamental questions in basic and translational research (e.g., how cell identity states and trajectories are determined in therapy resistance, metastasis formation, tissue development and somatic cell re-programming), potentially revealing new vulnerabilities that can serve as targets for therapies. Citation Format: Christian Umkehrer, Felix Holstein, Laura Formenti, Julian Jude, Kimon Froussios, Tobias Neumann, Shona M. Cronin, Lisa Haas, Jesse Lipp, Thomas R. Burkard, Michaela Fellner, Thomas Wiesner, Johannes Zuber, Anna C. Obenauf. CaTCH - A barcode-guided CRISPRa-inducible reporter to isolate clones from heterogeneous populations [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-132.

3048 – DELETION OF BAX: A NOVEL MECHANISM OF RESISTANCE TO BCL2 PATHWAY INHIBITORS IN AML CELL LINES

Acute myeloid leukemia (AML) is disease with poor outcomes, especially in older patients. Inhibition of apoptosis is a key mechanism by which leukemic cells proliferate and resist chemotherapy. Venetoclax (ABT199) is a selective inhibitor of the anti-apoptotic protein BCL2 that has been approved for use in combination with hypomethylating agents or with low dose cytarabine in elderly or unfit AML patients. The acquisition of resistance to ABT199 has been a cause of concern in venetoclax-based treatment approaches. Two major mechanisms of ABT199 resistance have been reported, namely: BCL2 mutations that affect drug binding (G101V, D103Y, etc.,) or over-expression of pro-survival proteins such as MCL-1 and BCL-xL. In order to study the mechanisms of acquired ABT199 resistance in AML, we created ABT199 resistant MV4-11 cells by treating the MV4-11 cells with incremental doses of ABT199. Five clones of ABT199 resistant cells, picked from semisolid media, were studied in detail. The resistant clones demonstrated a 160-350-fold higher resistance to ABT199 as compared to the parental MV4-11 cells. The resistant clones also demonstrated co-resistance to ABT-737 (BCL-2 and BCL-XL inhibitor), S63845 (MCL-1 inhibitor), and S55746 (BCL-2 inhibitor). There was no change in MCL-1 and BCL-XL at the protein level, however, there was a loss of BAX expression at RNA and protein levels in all resistant clones. Cytoscan HD arrays revealed a micro-deletion in the promoter region and the first 3 exons of the BAX gene in an ABT199 resistant clone. These deletions were further confirmed by performing genomic DNA PCR in all ABT199 resistant clones. In summary, we are reporting a novel mechanism of ABT199 resistance by genomic deletion of BAX in an AML cell line model. Loss of BAX renders the inhibitors of the BCL-2 pathway ineffective in inducing apoptosis. Alternative mechanisms of apoptosis induction need to be explored to overcome BAX deletion-induced resistance. BAX expression level/genomic status will be relevant in AML patients undergoing Venetoclax treatment.

Novel Genetic Mechanism of Venetoclax Resistance in AML: BAX Deletion

Venetoclax is a highly selective B-cell lymphoma 2 (BCL-2) inhibitor which recently received accelerated US FDA approval for use in combination with hypomethylating agents or with low-dose cytarabine in elderly or unfit acute myeloid leukemia (AML) patients. Venetoclax was also approved by the US FDA for the treatment of chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). Venetoclax-based AML treatment showed tolerable safety and favorable overall response rate in elderly patients with AML, particularly among high-risk groups, such as patients older than 75 yrs, adverse cytogenetics, and secondary AML. Acquisition of resistance to venetoclax in AML is the main cause of treatment failure in venetoclax-based AML treatment approaches. Intrinsic resistance in AML cells is attributed to increased levels of MCL-1 and/or BCL-XL, which lead to sequestration of pro-apoptotic proteins that are released from BCL-2 upon venetoclax treatment. Few reports of acquired resistance to venetoclax in other tumors describes acquisition of mutations in BCL-2 gene, for example: G101V in CLL and P104I in follicular lymphoma. With the increasing use of venetoclax in AML more reports of acquired and intrinsic resistance are expected, which in turn will shape future use of venetoclax. In order to study the mechanisms of acquired venetoclax resistance, we created venetoclax resistant MV4-11 cells by treating MV4-11 cells with incremental doses of venetoclax starting from 1nM to 100nM for a period of 8 weeks. Colonies of resistant MV4-11 clones were then isolated after culturing the cells in methylcellulose based medium. Five clones of venetoclax resistant cells (MV4-11 ABT-199R 1 through 5) were studied in detail. The MV4-11 ABT-199R clones demonstrated 300-700-fold higher resistance to venetoclax than the parental MV4-11 cells. The MV4-11 ABT-199R clones also demonstrated co-resistance to ABT-737 (BCL-2 and BCL-XL inhibitor), S63845 (MCL-1 inhibitor), and S55746 (BCL-2 inhibitor). Protein analysis by western blot demonstrated no differences in MCL-1 levels between resistant clones and the parental MV4-11 cells. Flow cytometry analysis demonstrated no significant change in the expression of BCL-XL in the resistant clones as compared to parental cells. Real-time PCR and western blotting showed loss of BAX expression. We subsequently performed whole-genome high density Cytoscan arrays for parental MV4-11 and one resistant clone. This revealed a micro-deletion in the resistant clone that included the promoter region and the first 3 exons of the BAX gene. These deletions were further confirmed by performing genomic DNA PCR. This was then further validated in the other 4 venetoclax resistant MV4-11 clones, showing similar genomic deletions of the region upstream of BAX and the first three exons of BAX gene. In summary, we are reporting a novel mechanism of venetoclax resistance by genomic deletion of BAX in an AML cell line. BAX is an essential gateway to mitochondrial apoptosis. As a result of BAX gene deletion, the inhibitors of BCL-2, BCL-XL, and MCL-1 are rendered ineffective in inducing apoptosis. Alternative mechanisms of apoptosis induction need to be explored to overcome BAX deletion-induced resistance. Disclosures No relevant conflicts of interest to declare.

Sleeping beauty transposon-mediated poly(A)-trapping and insertion mutagenesis in mouse embryonic stem cells.

Saturation mutagenesis of all endogenous genes within the mouse genome remains a challenging task, although a plenty of gene-editing approaches are available for this purpose. Here, a poly(A)-trap vector was generated for insertion mutagenesis in mouse embryonic stem (mES) cells. This vector contains an expression cassette of neomycin (Neo)-resistant gene lacking a poly(A) signal and flanked by two inverted terminal repeats of the Sleeping Beauty (SB) transposon. The whole poly(A)-trap cassette can transpose into target TA dinucleotides, properly splice with endogenous genes and effectively interrupt the transcription of trapped genes in mES cells after transient induction of SB expression by doxycycline (DOX)-treatment at 1 μg/ml, leading to the formation of multiple geneticin (G418)-resistant cell clones. In the first round of mutation screening, we identified six transposition events from 23 cell clones, including four inserted into an endogenous gene and two landed between endogenous genes. The abilities of self-renewal, totipotency, genetic stability and differentiation of syngap1+/- cells were not affected by DOX-treatment and G418-selection. These findings suggest that this SB transposon-mediated poly(A)-trap vector can be used as an alternative tool for a large-scale screening of mES cells with a gene mutation and for further generation of mutant mouse strains. Environ. Mol. Mutagen. 59:687-697, 2018. © 2018 Wiley Periodicals, Inc.

Time to tame necroptosis - viable combat against chemo resistant oral cancer cells.

Till 1998, a little was known about alternative forms of regulated cell death beside apoptosis. In present scenario, accumulating evidences suggest a form of programmed necrosis called Necroptosis which can be induced by various external stimuli including anticancer drugs, ionizing radiation, photodynamic therapy in the form of death domain receptor (DR) engagement by their respective ligands, TNF-alpha, Fas ligand (FasL) and TRAIL, under apoptosis deficient condition (caspase inhibitor), etc. receptor interacting protein-1 (RIP-1), a death domain containing kinase is the key molecule in necroptotic cell death pathway. On interaction with an additional protein RIP-3 to form an intracellular complex (complex-IIb), it triggers the various downstream mechanisms of necroptosis which includes: i) excessive production reactive oxygen species (ROS) as RIP-3 interacts with metabolic enzymes (glycogen phosphorylase, glutamate dehydrogenase) which increases the concentration of substrates for oxidative phophorylation - a major source of ROS; ii) mitochondrial dysfunction (mitrochondrial permeability transition ). Necrostatin (Nec-1) and CYLD act as negative and positive regulators for this mode of cell death.TNF the master pro-inflammatory cytokine has been known to either promote gene activation or to induce RIPK1 kinase-dependent cell death, in the form of apoptosis or necroptosis. Autophagy has also been proposed as an execution mechanism for necroptosis. There is growing evidence of impairment of necroptosis in tumerogenesis of various human cancers such as chronic lymphocytic leukemia, epidermal cancer and non Hodgkins lymphoma.As conventional anticancer drugs are usually apoptosis inducers, the development of apoptosis resistant cell clones is inevitable owing to cancer heterogeneity and mutation leading to failure of standard chemotherapy. It is a known fact that triggering necroptosis could be an alternative way to eradicate apoptosis-resistant cancer cells. Development of a new class of anticancer drug targeting this alternative pathway of the cell death is the need of the hour. Few in vitro and in vivo studies have been conducted showing excellent anti-tumor effect in both drug sensitive and resistant cases by targeting different modulators of necroptotic pathway: i) shikonin-a naturally occurring naphthoquinone showed prompt but profound anti-tumor effect on both primary and metastatic tumor i.e. cancer cell lines and osteosarcoma by inducing RIPK1 and RIPK3 dependent necroptosis; ii) staurosporine-generally accepted inducer of intrinsic apoptotic pathway and it is a wide spectrum inhibitor of protein kinases. It can induce necroptosis in caspase compromised conditions; iii) deoxypodophyllotoxin - a naturally occurring microtubule destabilizer successfully induced necroptosis in both drug sensitive and drug resistant cancer cell lines; iv) targeting Nec-1, a specific inhibitor of necroptosis can help in inducing necroptosis to enhance the radiosensitivity of cancer cells. Tanshinone IIA (Tan IIA) is known to induces both Nec-1 inhibition and FLIPS regulation-mediated apoptosis/necroptosis; v) obatoclax induces the interaction of p62 with RIP1K, RIP3K and FADD, key components of the necrosome and can mediate cell death in oral squamous cell carcinoma (OSCC) cells via autophagy-dependent necroptosis.Despite the rigorous implement of conventional therapies, increased number of refractory cases is unavoidable due to acquired resistance of cancer cells, badly affecting survival rate of OSCC. Additional knowledge about the mechanisms of cancer drug resistance and development of novel targeted therapy using alternative pathway of cell death and less susceptible to known resistance mechanisms i.e. necroptosis-based cancer therapy may help in designing effective anticancer strategies for OSCC .