Post-therapy Relapse Research Articles

Hepatic and Extrahepatic Characteristics of Autoimmune Hepatitis: A 23-year Hospital-Based Cohort Study.

The characteristics of autoimmune hepatitis (AIH) in Asia mostly remain elusive. A cohort study of liver biopsy-proven AIH patients was conducted in a tertiary care cancer of Taiwan. From 1999 to 2022, of 13,766 patients who underwent liver biopsy, 150 patients with AIH were enrolled. The female-to-male ratio was 2.26. At baseline, the mean age was 51.09 years, mean alanine aminotransferase level was 494.11 U/L, and 17 (11.3%) had cirrhosis. All except one patient had AIH type 1. The females were older and had higher baseline cirrhosis rates than did the males. The 23-year cumulative incidences of cirrhosis, hepatocellular carcinoma (HCC), mortality/liver transplantation, autoimmune diseases and extrahepatic cancer were 64.2%, 13.3%, 23.4%, 30.7% and 21.2%, respectively. The 1-year, 2-year, 3-year, 5-year, 10-year and 20-year postimmunosuppressive therapy relapse rates were 60%, 78.2%, 81.8%, 89.1%, 94.5% and 100%, respectively. Baseline associations were as follows: alkaline phosphatase (Alk-p) levels with postimmunosuppressive therapy flare [hazard ratio (HR): 1.003; 95% CI HR: 1.000-1.005]; age with HCC (1.072; 1.010-1.138) and all-cause cancer (1.041;1.005-1.079); cirrhosis with mortality/liver transplantation (11.933;1.984-71.787); and antinuclear antibody (ANA) titers with mortality/liver transplantation (1.001;1.000-1.003), cirrhosis (1.001;1.000-1.002), and autoimmune diseases (1.001; 1.000-1.002). In an Asian country endemic for viral hepatitis, the female-to-male and baseline cirrhosis rates of AIH patients were lower than expected, while over 60% of the patients eventually developed cirrhosis. The high posttherapy relapse rate warrants cautious monitoring, particularly for patients with high baseline Alk-p levels. Baseline age, cirrhosis status and ANA titers are crucial for outcomes.

Estrogen Receptor Alpha Mutations, Truncations, Heterodimers, and Therapies.

Annual breast cancer (BCa) deaths have declined since its apex in 1989 concomitant with widespread adoption of hormone therapies that target estrogen receptor alpha (ERα), the prominent nuclear receptor expressed in ∼80% of BCa. However, up to ∼50% of patients who are ER+ with high-risk disease experience post endocrine therapy relapse and metastasis to distant organs. The vast majority of BCa mortality occurs in this setting, highlighting the inadequacy of current therapies. Genomic abnormalities to ESR1, the gene encoding ERα, emerge under prolonged selective pressure to enable endocrine therapy resistance. These genetic lesions include focal gene amplifications, hotspot missense mutations in the ligand binding domain, truncations, fusions, and complex interactions with other nuclear receptors. Tumor cells utilize aberrant ERα activity to proliferate, spread, and evade therapy in BCa as well as other cancers. Cutting edge studies on ERα structural and transcriptional relationships are being harnessed to produce new therapies that have shown benefits in patients with ESR1 hotspot mutations. In this review we discuss the history of ERα, current research unlocking unknown aspects of ERα signaling including the structural basis for receptor antagonism, and future directions of ESR1 investigation. In addition, we discuss the development of endocrine therapies from their inception to present day and survey new avenues of drug development to improve pharmaceutical profiles, targeting, and efficacy.

Abstract 4013: Increasing mitochondrial respiration by silencing MCJ/DnaJC15 enhances CD8 CAR-T cell therapy efficacy

Abstract Chimeric antigen receptor (CAR)-T cell therapy is a promising treatment against leukemia and lymphoma. However, poor initial treatment response and post-therapy relapse have found to be common and remained to be resolved. Modulating T cell immunometabolism is considered a novel therapeutic approach to enhance CAR-T cell therapy. We have identified MCJ (DnaJC15) as an endogenous negative regulator of Complex I of the electron transport chain in mitochondria. MCJ is abundantly expressed in mouse CD8 cells relative to other immune cells. We propose that MCJ can be a therapeutic target to enhance mitochondrial respiration in CD8 CAR-T cells. We will show that loss of MCJ in mouse CD8 cells leads to superior anti-tumor response against melanoma. In addition, we will show that MCJ-deficient murine CD8 CAR-T cells have enhanced killing activity against leukemia both in vitro and in vivo. We have recently revealed that MCJ is also expressed in human CD8 cells and the levels of MCJ are heterogenous among human population. Similar to murine CD8 cells, MCJ knockdown in human CD8 cells enhances mitochondrial membrane potential and effector functions. We have designed a new CAR construct to silence MCJ in human CD8 CAR-T cells. MCJ-deficient human CD8 cells show superior cytotoxicity against leukemia with increased mitochondrial metabolism. Together, these data demonstrate that MCJ is a promising target to improve CAR-T cell therapy through modulating the T cell metabolism. We are currently investigating the effect of MCJ-deficient CD8 CAR-T cells against solid malignancy. Citation Format: Meng-Han Wu, Emily L. Giddings, Felipe Valenca-Pereira, Catherine Pham-Danis, Francesca Cendali, Michael C. Yarnell, Amanda J. Novak, Jorge Henao-Mejia, Richard A. Flavell, Angelo D'Alessandro, Mark Eric Kohler, Mercedes Rincon. Increasing mitochondrial respiration by silencing MCJ/DnaJC15 enhances CD8 CAR-T cell therapy efficacy [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 4013.

Transcriptomes of the tumor-adjacent normal tissues are more informative than tumors in predicting recurrence in colorectal cancer patients

BackgroundPrevious investigations of transcriptomic signatures of cancer patient survival and post-therapy relapse have focused on tumor tissue. In contrast, here we show that in colorectal cancer (CRC) transcriptomes derived from normal tissues adjacent to tumors (NATs) are better predictors of relapse.ResultsUsing the transcriptomes of paired tumor and NAT specimens from 80 Korean CRC patients retrospectively determined to be in recurrence or nonrecurrence states, we found that, when comparing recurrent with nonrecurrent samples, NATs exhibit a greater number of differentially expressed genes (DEGs) than tumors. Training two prognostic elastic net-based machine learning models—NAT-based and tumor-based in our Samsung Medical Center (SMC) cohort, we found that NAT-based model performed better in predicting the survival when the model was applied to the tumor-derived transcriptomes of an independent cohort of 450 COAD patients in TCGA. Furthermore, compositions of tumor-infiltrating immune cells in NATs were found to have better prognostic capability than in tumors. We also confirmed through Cox regression analysis that in both SMC-CRC as well as in TCGA-COAD cohorts, a greater proportion of genes exhibited significant hazard ratio when NAT-derived transcriptome was used compared to when tumor-derived transcriptome was used.ConclusionsTaken together, our results strongly suggest that NAT-derived transcriptomes and immune cell composition of CRC are better predictors of patient survival and tumor recurrence than the primary tumor.

Citrus aurantium Peel Extract Combined with Doxorubicin-Induced ROS-driven Cell Cycle Arrest and Apoptosis of Triple-Negative Breast Cancer Cells

The current approach to breast cancer has problems with the emergence of resistance, side effects, and even the emergence of post-therapy relapses. One of the reasons is that the available chemotherapy is still based on cytotoxicity through cell cycle inhibition and apoptosis induction. In fact, there are still several mechanisms for the direction of cytotoxicity to become more prospective targets of chemotherapy action, such as reactive oxygen species (ROS) leading to cell death induction. One prospective candidate from natural ingredients is Citrus aurantium peel extract (CSP). This study aims to develop a CSP as a co-chemotherapy candidate that leads to aging induction and ROS modulation in breast cancer cells. The breast cancer cell model used is triple-negative breast cancer cells (TNBC), which is a highly metastatic cell model. Apoptosis and cell cycle modulation profiles were analyzed under PI-Annexin and PI flow cytometry, respectively. The ROS level was evaluated under DCFDA flow cytometry. The combination of CSP and Dox induces oxidative stress with ROS levels up to 3.5 times. The increase in ROS levels was in line with the dose-dependent induction of apoptosis and induced G2/M phase cell cycle arrest. Taken together, CSP potentially induces the Dox effect on MDA-MB-231 cells, which may be mediated by the elevation of the ROS levels leading to cell death induction.

Abstract 1863: Mouse strain variations in drug-induced lung stromal responses

Abstract Chemotherapy has significantly improved the survival of cancer patients. However, the vast variation in treatment responses remains a major issue in the clinic. It is imperative to understand the molecular mechanisms underlying treatment failure in specific populations of patients. Genetic diversity has been recognized to mainly account for the differences in drug responses among patients. Yet, it is not feasible to map the responsible genetic variants within millions of single-nucleotide polymorphisms in the human genome in clinical settings with a limited number of subjects and insufficient genomic information. Genetically diverse mice are an ideal tool to overcome the limitations in clinical studies and model drug responses in hosts that require genetic susceptibility and resistance factors. Recent studies have also implicated a critical role for host cells (i.e., the tissue microenvironment) in building a protective “niche” for tumor cells enabling their escape from chemotherapeutic treatments. Notably, the host regenerative response upon chemotherapy “injury,” which is regarded as an intrinsic host mechanism to repair damaged tissues, may be exploited by tumor cells for their local recurrence or distant metastases. In this study, using a lung injury model, we determined how drug-induced lung stromal wound healing responses differed among eight inbred mouse strains (BALB/cJ, NSG, C57BL/6J, CH3/HeJ, CBA/J, SJL/J, A/J, and NOD/ShiLtJ). Upon either cisplatin or doxorubicin stimulation, the wound healing-associated genes Il-6, Spp1, Cxcl1, and Ccl2 were all found upregulated in lung stromal cells but showed a great variation across different strains. Furthermore, we performed single-cell RNA sequencing on the lung stromal cells and immune cells isolated from three selected mouse strains (BALB/cJ, C57BL/6J, and NOD/ShiLtJ) without and with doxorubicin treatment in vivo. The transcriptomic analyses revealed strain-specific and non-specific therapy-elicited gene signatures in different types of lung tissue cells. Collectively, our results signify an association between genetic backgrounds and drug-induced host responses in mice. The next steps of the study aim to understand how genetic diversity would functionally impact drug-induced lung tissue regeneration, lung inflammation, and post-therapy tumor relapse or metastasis in the lung. Citation Format: Carlos M. Leon, Zheng Gong, Qing Li, Jiayuan Shi, Wulin Zuo, Muneer G. Hasham, Lenny Shultz, Sheng Li, Guangwen Ren. Mouse strain variations in drug-induced lung stromal responses [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 1863.

Abstract 3749: Mapping the DNA accessibility landscape of BCP-ALL patients revealed principles of tumor evolution

Abstract Pediatric B-Acute Lymphoblastic Leukemia (B-ALL) is the primary cause of death from hematological disease in children. Despite the enormous improvement of treatments based on innovative immunotherapy, applications of new approaches constitute a significant emergency in clinical practice. We have built the most extensive map of the accessibility landscape of 35 B-Cell Precursor ALL (BCP-ALL) to date. We integrated this map with a plethora of transcriptomic and epigenomics pan-cancer profiles to define the key determinant of BCP-ALL post-therapy relapse. We show that chromatin accessibility dynamics play a major regulatory role in BCP-ALL heterogeneity and sustain disease evolution. We have analyzed a large cohort of tissues from healthy donors (N=6) and BCP-ALL patients at time of Primary onset (N=11), Remission (N=7),or Relapse (N=9. We generated genome-wide accessibility profiles using the Assay for Transposase- Accessible Chromatin (ATAC-seq) at the coverage of 50-60 million reads of each sample. We observed that regulatory elements are strongly associated with epigenetic heterogeneity, but dominant elements have a higher chance of being shared across patients. By differential analysis of chromatin layout between healthy profiles and primary tissues, we identified 688 regulatory regions being deactivated and 758 activated during cancer early stages. Interestingly, novel activated sites recapitulate B-ALL characteristics interrogating the Disease Ontology database and gene ontology (Pval< 10-5). Relapsed patients are dominated by regulatory elements (N=~6000)originally represented at diagnosis that shrinks undertreatments and subsequently re-expand, driving the relapse. HiC maps, H3K27ac, and H3K4me1 ChIP-seq experiments integrated with chromatin maps obtained from ENCODE, GTEX, TCGA, and transcriptomic data generated by the TARGET project confirm that a large percentage of these elements are high fidelity active enhancers. The activity of these enhancers increases the risk of relapse significantly. Particularly, DCTD, MYB, and BCL2 genes are actively regulated by a set of plastic enhancer elements characterized with experimental and computational approaches. We gained insights into the role ofthe DCTD gene regulation using CRISPR-Cas9-mediated deletion of the DCTD-enhancer, leading to decreased level of DCTD gene mRNA of 40% accompanied by acell proliferation reduction of 30%. Next, we characterized the cistrome landscape of our patient cohort at accessible sites relative to the disease status. Foot printing analysis suggested ERG and EBF transcription factors (TFs) as a fundamental player in sustaining the activity of the most dynamical enhancers. In conclusion, chromatin accessibility shifts can be used to trace phenotypic changes throughout the journey of BCP-ALL patients and provide novel insights in targeting cancer phenotypical modulators. Citation Format: Giacomo Corleone, Cristina Sorino, Matteo Caforio, Stefano Di Giovenale, Francesca De Nicola, Valentina Bertaina, Angela Pitisci, Franco Locatelli, Maurizio Fanciulli, Valentina Folgiero. Mapping the DNA accessibility landscape of BCP-ALL patients revealed principles of tumor evolution [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 3749.

EFFICACY AND SAFETY OF EXPERIMENTAL CHIMERIC ANTIGEN RECEPTOR (CAR) T-CELLS VERSUS AXICABTAGENE CILOLEUCEL (YESCARTA) FOR THE TREATMENT OF RELAPSED/REFRACTORY LARGE B-CELL LYMPHOMA: MATCHING ADJUSTED INDIRECT COMPARISONS AND SYSTEMATIC REVIEW

Despite the high response rates seen with Chimeric Antigen Receptor (CAR) T-cell therapy for relapsed/refractory Large B-cell lymphoma (R/R LBCL), post-therapy relapse remains a key challenge. To date, no study has evaluated the comparative efficacy and safety of experimental CAR T-cell products versus currently approved CAR T-cell therapies. To indirectly compare the efficacy and safety of novel, experimental CAR T-cell products against the first FDA-approved CAR T-cell construct, Axicabtagene ciloleucel (Yescarta). In compliance with the PRISMA guidelines, we performed a systematic review, which identified 16 independent, early-phase clinical trials consisting of 193 LBCL patients with individual patient data (IPD). We categorized eight pooled populations based on the target antigens (CD19, CD20), co-stimulatory domains (CD28, 4-1BB), and CAR T-cells administered with or without concomitant autologous stem cell transplant (ASCT). The pooled populations were categorized as follows: (1) dual targeting strategies, such as tandem CD19.CD20, n = 28; (2) co-infusion of CD19 and CD20 CARs, n = 21; (3) third-generation CARs, n = 26; (4) CD19 CARs with modified constructs for reduced toxicity, including CD19.BBz.86, n = 21; and (5) Hu19.CD828Z, n = 14; (6) CD19. 4-1BB.S manufactured in China, n = 24; (7) concomitant ASCT and CD19.CD28, n = 45; and (8) CD20 CARs with a 4-1BB co-stimulatory domain, n = 14. A Matching Adjusted Indirect Comparison (MAIC) statistical technique was applied to account for heterogeneity in the study population across trials. Estimates for the experimental CAR T-cell trials were adjusted using patient-level data to match the ZUMA-1 (Yescarta, n = 108) study population based on mutually reported key baseline covariates, including age, disease stage, histology, refractoriness, number of prior lines of therapy, and extranodal disease. The study endpoints for this study included progression-free survival (PFS), cytokine release syndrome (CRS), and neurotoxicity (NT). In the dual-targeting strategy, only tandem CD19.CD20.4-1BB was associated with a statistically significantly improved PFS (HR = 0.46; 95% CI, 0.23-0.92) and a safer NT profile (OR = 0.15; 95% CI, 0.03-0.76) compared to Yescarta. As for safety, significantly reduced NT (OR = 0.19; 95% CI, 0.04-0.94) was also noted with third-generation CAR T-cells. None of the other comparisons were statistically significant. Of note, no statistically significant associations regarding PFS or safety were seen with the sequential administration of ASCT followed by CAR T-cells within 4-7 days. CD19. BBz.86 and Hu19. CD828Z CAR T-cell products containing modified co-stimulatory domains aimed at reduced toxicity demonstrated no grade ≥3 CRS nor NT occurrence. Anti-CD20.4-1BB did not show a remarkable difference in terms of the selected endpoints for this study. Safety data for the co-infusion of CD19 and CD20 were not available for this trial. Our MAIC suggests a dual targeting approach using tandem CD19.CD20.4-1BB might have both superior efficacy and safety compared to Yescarta. Future studies comparing experimental CAR T-cell constructs with other CD19. 4-1BB-based approved CAR T-cell products, such as Tisagenlecleucel (Kymriah) and Lisocabtagene maraleucel (Breyanzi), may further contribute to clarify these findings.

Descriptive and Functional Genomics in Acute Myeloid Leukemia (AML): Paving the Road for a Cure.

Simple SummaryAcute myeloid leukemia (AML) accounts for 7.6% of hematopoietic malignancies with a long-term survival of less than 20%. Better understanding its physiopathology and finding new treatments remain important issues. In the current review, the authors discuss how genetic engineering technologies improvement allowed a better genetic characterization of AML. Such molecular dissection of the AML genome had two direct clinical impacts: a prognostic contribution by defining a new molecular classification of AML which guides therapeutic regimen intensity, and a therapeutic impact by allowing the identification of new therapeutic targets. New genome editing tools and animal models have also paved the way for a better understanding of AML leukemogenesis. Their impact is also summarized in this review. The combination of descriptive and functional genetics may ultimately be the key to improving the prognosis of this dismal disease.Over the past decades, genetic advances have allowed a more precise molecular characterization of AML with the identification of novel oncogenes and tumor suppressors as part of a comprehensive AML molecular landscape. Recent advances in genetic sequencing tools also enabled a better understanding of AML leukemogenesis from the preleukemic state to posttherapy relapse. These advances resulted in direct clinical implications with the definition of molecular prognosis classifications, the development of treatment recommendations based on minimal residual disease (MRD) measurement and the discovery of novel targeted therapies, ultimately improving AML patients’ overall survival. The more recent development of functional genomic studies, pushed by novel molecular biology technologies (short hairpin RNA (shRNA) and CRISPR-Cas9) and bioinformatics tools design on one hand, along with the engineering of humanized physiologically relevant animal models on the other hand, have opened a new genomics era resulting in a greater knowledge of AML physiopathology. Combining descriptive and functional genomics will undoubtedly open the road for an AML cure within the next decades.

Combination of a mitogen-activated protein kinase inhibitor with the tyrosine kinase inhibitor pacritinib combats cell adhesion-based residual disease and prevents re-expansion of FLT3-ITD acute myeloid leukaemia.

Minimal residual disease (MRD) in acute myeloid leukaemia (AML) poses a major challenge due to drug insensitivity and high risk of relapse. Intensification of chemotherapy and stem cell transplantation are often pivoted on MRD status. Relapse rates are high even with the integration of first-generation FMS-like tyrosine kinase 3 (FLT3) inhibitors in pre- and post-transplant regimes and as maintenance in FLT3-mutated AML. Pre-clinical progress is hampered by the lack of suitable modelling of residual disease and post-therapy relapse. In the present study, we investigated the nature of pro-survival signalling in primary residual tyrosine kinase inhibitor (TKI)-treated AML cells adherent to stroma and further determined their drug sensitivity in order to inform rational future therapy combinations. Using a primary human leukaemia-human stroma model to mimic the cell-cell interactions occurring in patients, the ability of several TKIs in clinical use, to abrogate stroma-driven leukaemic signalling was determined, and a synergistic combination with a mitogen-activated protein kinase (MEK) inhibitor identified for potential therapeutic application in the MRD setting. The findings reveal a common mechanism of stroma-mediated resistance that may be independent of mutational status but can be targeted through rational drug design, to eradicate MRD and reduce treatment-related toxicity.

The mitochondrial citrate carrier, SLC25A1, drives stemness and therapy resistance in non-small cell lung cancer

Therapy resistance represents a clinical challenge for advanced non-small cell lung cancer (NSCLC), which still remains an incurable disease. There is growing evidence that cancer-initiating or cancer stem cells (CSCs) provide a reservoir of slow-growing dormant populations of cells with tumor-initiating and unlimited self-renewal ability that are left behind by conventional therapies reigniting post-therapy relapse and metastatic dissemination. The metabolic pathways required for the expansion of CSCs are incompletely defined, but their understanding will likely open new therapeutic opportunities. We show here that lung CSCs rely upon oxidative phosphorylation for energy production and survival through the activity of the mitochondrial citrate transporter, SLC25A1. We demonstrate that SLC25A1 plays a key role in maintaining the mitochondrial pool of citrate and redox balance in CSCs, whereas its inhibition leads to reactive oxygen species build-up thereby inhibiting the self-renewal capability of CSCs. Moreover, in different patient-derived tumors, resistance to cisplatin or to epidermal growth factor receptor (EGFR) inhibitor treatment is acquired through SLC25A1-mediated implementation of mitochondrial activity and induction of a stemness phenotype. Hence, a newly identified specific SLC25A1 inhibitor is synthetic lethal with cisplatin or with EGFR inhibitor co-treatment and restores antitumor responses to these agents in vitro and in animal models. These data have potential clinical implications in that they unravel a metabolic vulnerability of drug-resistant lung CSCs, identify a novel SLC25A1 inhibitor and, lastly, provide the first line of evidence that drugs, which block SLC25A1 activity, when employed in combination with selected conventional antitumor agents, lead to a therapeutic benefit.

Diagnostic Utility of 18F-FDG PECT/CT in Assessment of Post-therapy Remission or Relapse of Lymphoma

Background: Treatment and prognosis of lymphoma depend on accurate staging and evaluation. Relapsed Lymphomas represent great problem in accurate quick diagnosis that imply appropriate treatment. Aim of the work: To evaluate diagnostic utility of 18F-FDG PECT/CT in assessment of treatment response and identification of disease relapse. Patients and Methods: This prospective study were conducted on 49 consecutive patients with histologically verified lymphoma. They were 27/49 (55.1%) male and 22/49 (44.9%) female aged 37.83±2.29. All patients were subjected to pretreatment as well a follow up clinical, laboratory, CT PET-CT. PET-CT findings were classified as positive and negative according to remission status and relapse. The sensitivity, specificity and accuracy of PET- CT in assessment of the remission status and relapse were calculated and to be confirmed by open or laparoscopic biopsy. Results: PET-CT detected complete regression in 28 patients (57.14%), partial regression in 4 patients (8.16%), stationary course in 2 patients (4.08%), progression / relapse in 15 patients (30.61%). According to PET/CT 9 (23%) patients were true positive, 25 (65%) were true negative and 5 (13%) were false negative with sensitivity, specificity, positive p predictive value and negative predictive value of 95% and 91%, 90% and 98%, respectively. Conclusion: Our results confirm FDG-PET as a valid tool for assessment of treatment response and detection of relapsing lymphoma. We recommended further multicentric prospective studies that incorporates a larger number of patients should be implemented to define the exact relation between certain clinical, pathological, laboratory and PET factors and fate of the disease.

ASXL2 Is Recurrently Mutated in t(8;21) AML and Regulates Hematopoietic Development

Chromosomal translocation t(8;21) (q22;q22) leading to generation of oncogenic RUNX1-RUNX1T1 fusion is a cytogenetic abnormality observed in about 10% of acute myelogenous leukemia (AML). Studies in animal models and recent next generation sequencing approaches have suggested cooperativity of secondary genetic lesions with t(8;21) in inducing leukemogenesis. In this study, we used targeted and whole exome sequencing of 93 cases (including 30 with matched relapse samples) to profile the mutational landscape of t(8;21) AML at initial diagnosis and post-therapy relapse. We identified recurrent mutations of KIT, TET2, MGA, FLT3, NRAS, DHX15, ASXL1 and KMT2Dgenes in this subtype of AML. In addition, high frequency of truncating alterations in ASXL2 gene (19%) also occurred in our cohort. ASXL2 is a member of mammalian ASXL family involved in epigenetic regulation through recruitment of polycomb or trithorax complexes. Unlike its closely related homolog ASXL1, which is mutated in several hematological malignancies including AML, MDS, MPN and others; mutations of ASXL2 occur specifically in t(8;21) AML.We observed that lentiviral shRNA-mediated silencing of ASXL2 impaired in vitro differentiation of t(8;21) AML cell line, Kasumi-1, and enhanced its colony forming ability. Gene expression analysis uncovered dysregulated expression of several key hematopoiesis genes such as IKZF2, JAG1, TAL1 and ARID5B in ASXL2 knockdown Kasumi-1 cells. Further, to investigate implications of loss of ASXL2 in vivo, we examined hematopoiesis in Asxl2 deficient mice. We observed an age-dependent increase in white blood cell count in the peripheral blood of Asxl2 KO mice. Myeloid progenitors from Asxl2 deficient mice possessed higher re-plating ability and displayed altered differentiation potential in vitro. Flow cytometric analysis of >1 year old mice revealed increased proportion of Lin-Sca1+Kit+ (LSK) cells in the bone marrow of Asxl2 deficient mice, while the overall bone marrow cellularity was significantly reduced. In vivo 5-bromo-2’-deoxyuridine incorporation assay showed increased cycling of LSK cells in mice lacking Asxl2. Asxl2 deficiency also led to perturbed maturation of myeloid and erythroid precursors in the bone marrow, which resulted in altered proportions of mature myeloid populations in spleen and peripheral blood. Further, splenomegaly was observed in old ASXL2 KO mice and histological and flow cytometric examination of ASXL2 deficient spleens demonstrated increased extramedullary hematopoiesis and myeloproliferation compared with the wild-type controls. Surprisingly, loss of ASXL2 also led to impaired T cell development as indicated by severe block in maturation of CD4-CD8- double negative (DN) population in mice >1 year old. These findings established a critical role of Asxl2 in maintaining steady state hematopoiesis. To gain mechanistic insights into its role during hematopoietic differentiation, we investigated changes in histone marks and gene expression affected by loss of Asxl2. Whole transcriptome sequencing of LSK population revealed dysregulated expression of key myeloid-specific genes including Mpo, Ltf, Ngp Ctsg, Camp and Csf1rin cells lacking Asxl2 compared to wild-type control. Asxl2 deficiency also caused changes in histone modifications, specifically H3K27 trimethylation levels were decreased and H2AK119 ubiquitination levels were increased in Asxl2 KO bone marrow cells. Global changes in histone marks in control and Asxl2 deficient mice are being investigated using ChIP-Sequencing. Finally, to examine cooperativity between the loss of Asxl2 and RUNX1-RUNX1T1 in leukemogenesis, KO and wild-type fetal liver cells were transduced with retrovirus expressing AML1-ETO 9a oncogene and transplanted into irradiated recipient mice, the results of this ongoing study will be discussed.Overall, our sequencing studies have identified ASXL2 as a gene frequently altered in t(8;21) AML. Functional studies in mouse model reveal that loss of ASXL2 causes defects in hematopoietic differentiation and leads to myeloproliferation, suggesting an essential role of ASXL2 in normal and malignant hematopoiesis.*LH and NH contributed equally DisclosuresOgawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

Abstract LB-229: Novel mitochondrial inhibitors hold promise for the eradication of lung cancer stem cells

Abstract Lung cancer therapy failure is attributed not only to the genetic heterogeneity of tumors, but is also promoted by the functional and phenotypic plasticity of different cell populations. The cancer stem cell (CSC) theory postulates that an under-represented population of cells with different growth- and drug-sensitivity properties is responsible for metastatic dissemination and post-therapy relapse. The detailed molecular and phenotypical analysis of CSCs in primary human lung cancers has been limited however, because CSCs have poor proliferative capacity when placed in culture and eventually undergo terminal differentiation. We have developed a novel culturing system that permits the rapid expansion of patient-derived cancer samples and is allowing us to rapidly build a collection of non-small-cell (NSCLC) lung cancers for real-time drug screening as well as for molecular and phenotypical characterization. Compared to other known culturing conditions, this system leads to a stark enrichment of CSCs, defined as such based on the expression of stem cell markers, the ability to differentiate towards multiple lineages and to form tumors at limiting dilutions in immuno-deficient mice. Because CSCs tend to be refractory to targeted or untargeted therapies, we have studied these CSC populations with respect to their metabolic vulnerabilities. CSCs reside in stem cell niches that are deprived of nutrients and of glucose and are characterized by low radical oxygen species (ROS) levels, thus likely relying upon different metabolic requirements relative to other cancer cells which represent the “bulk” of the tumor mass. We found that CSCs populations derived from tumors harboring diverse mutational profiles share, as a common feature, the reliance upon mitochondrial respiration for growth and expansion. We show that this metabolic aberration is sustained, for the most part, by the mitochondrial citrate transporter SLC25A1, whose expression levels are elevated in NSCLC predicting poorest prognostic outcome. SLC25A1 is required for the expansion of CSCs where it promotes the homeostatic energetic output of the mitochondria via oxidative phosphorylation, while overcoming the glycolytic addition, thus essentially reverting the Warburg effect which has been thus far considered a hallmark of tumor cells. We have also developed two chemical inhibitors of SLC25A1 activity and we demonstrate that these inhibitors target lung CSCs by blocking mitochondrial respiratory activity, by inducing mitochondrial dysfunction and by altering the redox environment. These alterations lead to energetic crisis and to cell death. These agents show remarkable anti-tumor activity as single agents in xenograft experiments in mice and they enhance sensitivity to platinum-based chemotherapy. In conclusion we have identified a novel class of agents that specifically targets the mitochondrial metabolism of cancer stem cells. We propose that such activity may permit eradication of the cell population primarily responsible of drug resistance and metastatic dissemination regardless of the mutational status of the tumor of origin. These findings could set the ground for clinical trials employing mitochondrial inhibitors as a rescue-treatment after failure with currently available targeted or untargeted therapies. Citation Format: Harvey Fernandez, Shreyas Grade, Kyu Ah Kim, Mayalin Laforteza, Yuwen Zhang, Chris Albanese, Mikell Paige, Giuseppe Giaccone, Maria L. Avantaggiati. Novel mitochondrial inhibitors hold promise for the eradication of lung cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-229.

Cancer stem cell markers: premises and prospects.

Over the last decade, compelling evidence has shown that cancer stem cells (CSCs) exist in a variety of malignancies. The conventional method for anticancer therapy involves targeting only the proliferating mitotic cells, sparing the slow-cycling cells that eventually evade chemotherapy and become a source of post therapy relapses. With the increasing awareness of CSCs supported by sophisticated experimental evidence, therapeutic strategies today are aimed at selectively identifying and targeting CSCs using biomarkers. The ability to identify CSCs allows targeted elimination of these cancer-initiating cells. Herein, we discuss CSC markers in the context of different types of cancers, their significance in selectively identifying CSCs and the therapeutic implications of using these biomarkers to prevent invasion and metastasis of cancer.

P34: Plasma micro-RNAs as noninvasive biomarkers for diagnostics of lung cancer

Lung cancer (LC) is currently the world’s leading cause of cancer-related mortality with overall mortality to incidence ratio of 0.87. Only 15% of LC patients are diagnosed at early stage of disease and have 5-year survival rate of 54%, whereas 56% of patients have distant metastases and 5-year survival rate of about 4%. Current methods of lung cancer diagnostics are not efficient as screening tools due to high costs (CT), low sensitivity and health risks (radiology), invasiveness (biopsy) or low prevalence in population (genetic alterations). New strategies for preclinical lung cancer screening as well as monitoring of post therapy relapses are required. MiRNAs circulating in blood were shown to reflect the progression of disease and are thus considered as potential biomarkers for cancer diagnostics and theranostics. In this study we investigated seven-miRNA signature circulating in plasma of lung cancer patients and healthy individuals. All seven miRNAs were previously shown to be involved in either regulation of cell cycle and apoptosis and/or tumor development, invasion and vascularization. Blood samples of 50 healthy individuals were obtained from Center of New Medical Technologies (Novosibirsk, Russia) and Novosibirsk Research Institute of Circulation Pathology of E.N. Meshalkin (Novosibirsk, Russia). Samples of 75 lung cancer patients, including 52 patients with squamous cell carcinoma (SCC) and 18 patients with adenocarcinoma (AC) were obtained from Novosibirsk Research Institute of Circulation Pathology of E.N. Meshalkin (Novosibirsk, Russia) and Tomsk Cancer Research Institute RAMS (Tomsk, Russia). None of the patients have undergone surgical treatment or received chemotherapy prior to blood collection. Lung biopsy specimens and imaging techniques were applied to confirm the histopathological features and tumor stages of LC patients. Study was approved by ethical committees of all participating organizations and written informed consent was provided by all participants. Circulating miRNAs were isolated from blood plasma using a single-phase phenol-free protocol (Zaporozhchenko et al., Anal Biochem, 2015; Rus. patent application No. 2014137763, priority date 17.09.2014). Concentrations of miRNAs (miR-21, miR-19b, miR-126, miR-25, miR-205, miR-183, miR-125b) were measured by qRT-PCR and normalized to miR-16. Concentrations of four miRNAs (miR-19b, miR-21, miR-25, miR-183) were significantly different in lung cancer versus healthy individuals (p Receiver Operating Characteristic (ROC) curve analysis of differentially expressed miRNAs in total study population and subtype-based groups showed that miR-19b has highest predictive value for total population and SCC patients, while miR-183 was more effective in discriminating patients with AC. All miRNAs except miR-21 showed strong bias towards one of the subtypes. Presumably a combination of miRNAs with opposite bias should provide a more potent diagnostic tool for cancer detection in total population than individual miRNAs or panels of miRNAs that are specific to one cancer subtype. Indeed, stepwise binary logistic regression has identified the combination of miR-19b and miR-183 to be a strong prediction of disease in total population and yielded a solid increase in AUC: 0,990 (miR-19b+miR-183) versus 0,806 (miR-19b) or 0,924 (miR-183). This combination can be used to identify lung cancer with 94.7% sensitivity and 95.2% specificity. Thus, biological aspects such as tumor genetics and phenotype, stage of disease, response to therapy, and other meaningful tumor properties of such heterogeneous malignancies as LC can interfere with contents and composition of cell-free miRNA pool. Wide profiling of circulating miRNAs will specify biomarkers of LC phenotypes and improve non/less-invasive LC diagnostics.

Conditional Expression of a Leukemogenic Fusion Oncogene in Murine Hematopoietic Stem Cells Results in Highly Invasive AML with Origin-Related Genetic Signatures of Prognostic Significance for the Human Disease

Acute myeloid leukemia (AML) is a genetically and clinically heterogeneous disease. Chromosomal translocations causing fusions of the Mixed Lineage Leukemia (MLL) gene are associated with pediatric and adult de novo and therapy-related acute leukemia that are characterized by variable disease outcome. To date, only a limited number of genetic lesions have been implicated in AML disease variability. To address the impact of cellular origin on disease heterogeneity of AMLs, we studied AMLs originating from long-term hematopoietic stem cells (LT-HSCs) and more committed progenitors using a newly established inducible “iMLL-AF9” transgenic mouse model for the t(9:11)(p22;q23) translocation associated MLL-AF9 oncogene. Ex vivo immortalized cells displayed several origin-related growth and drug resistance characteristics and gene expression signatures. Only iMLL-AF9 expressing LT-HSCs formed novel, particularly dispersed colonies, expanded in lineage restrictive stem cell medium and were resistant to genotoxic stress. iMLL-AF9 induction in vivo resulted in fully reversible myelo-monoblastic AML in all animals. Intriguingly, induction of iMLL-AF9 in LT-HSCs caused a particularly aggressive AML phenotype in 15% of recipient mice while the remainder LT-HSCs, as well as short-term HSCs, common myeloid and granulocyte macrophage progenitors induced a more moderate AML. The aggressive LT-HSC-derived AMLs were all characterized by a drastically shorter latency (37 versus 72 days median latency), higher white blood counts, increased invasion capacity and chemo-resistance of leukemic blast, and were associated with expression of genes previously implicated in cell migration, invasion, inflammation and the epithelial-mesenchymal transition (EMT) of solid cancers. shRNA based knock-down experiments demonstrated functional importance of selected candidate genes in cell migration and invasion. Importantly, comparative gene expression analyses between mouse and human revealed that among the genes associated with aggressive AMLs in mice, elevated expression of 66, 11 and 40 human orthologous genes was significantly associated with poor overall survival of t(9;11) (n=21), 11q23-lesion positive, (n=54) and all AMLs (n=662) (p<0.05). Collectively, our data indicates that expression of MLL-AF9 in HSCs results in a particularly aggressive disease driven by expression of common MLL targets and origin-dependent targets previously associated with migration, invasion and EMT of aggressive solid cancers. Remarkably, origin-related genetic signatures associated with the aggressive murine disease revealed a large number of novel MLL-AF9 fusion targets and many highly significant genetic prognostic markers for the overall survival in human AML irrespective of the underlying genetic alterations. Our data experimentally support the previously disputed theory that human AML may also arise from stem and/or oligo-potent progenitors contributing thus to the great heterogeneity of AML including drug resistance and post therapy relapse. Validation of the novel identified target genes in a broader spectrum of human leukemia will facilitate the design of accurate personalized therapeutic interventions. DisclosuresNo relevant conflicts of interest to declare.

Efficacy of NZ2114, a Novel Plectasin-Derived Cationic Antimicrobial Peptide Antibiotic, in Experimental Endocarditis Due to Methicillin-Resistant Staphylococcus aureus

Cationic antimicrobial peptides (CAPs) play important roles in host immune defenses. Plectasin is a defensin-like CAP isolated from the saprophytic fungus Pseudoplectania nigrella. NZ2114 is a novel variant of plectasin with potent activity against Gram-positive bacteria. In this study, we investigated (i) the in vivo pharmacokinetic and pharmacodynamic (PK/PD) characteristics of NZ2114 and (ii) the in vivo efficacy of NZ2114 in comparison with those of two conventional antibiotics, vancomycin or daptomycin, in an experimental rabbit infective endocarditis (IE) model due to a methicillin-resistant Staphylococcus aureus (MRSA) strain (ATCC 33591). All NZ2114 regimens (5, 10, and 20 mg/kg of body weight, intravenously [i.v.], twice daily for 3 days) significantly decreased MRSA densities in cardiac vegetations, kidneys, and spleen versus those in untreated controls, except in one scenario (5 mg/kg, splenic MRSA counts). The efficacy of NZ2114 was clearly dose dependent in all target tissues. At 20 mg/kg, NZ2114 showed a significantly greater efficacy than vancomycin (P < 0.001) and an efficacy similar to that of daptomycin. Of importance, only NZ2114 (in 10- and 20-mg/kg regimens) prevented posttherapy relapse in cardiac vegetations, kidneys, and spleen, while bacterial counts in these target tissues continued to increase in vancomycin- and daptomycin-treated animals. These in vivo efficacies were equivalent and significantly correlated with three PK indices investigated: fC(max)/MIC (the maximum concentration of the free, unbound fraction of a drug in serum divided by the MIC), fAUC/MIC (where AUC is the area under the concentration-time curve), and f%T(>MIC) (%T(>MIC) is the cumulative percentage of a 24-h period that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions), as analyzed by a sigmoid maximum-effect (E(max)) model (R(2) > 0.69). The superior efficacy of NZ2114 in this MRSA IE model suggests the potential for further development of this compound for treating serious MRSA infections.

Radiotherapy and risks of tumor regrowth or inducing second cancer.

Considerable research is aimed at determining the mechanism by which tumor cures, or regrows or second cancer develops, to be predictable and controllable. The wide range of doses, from low to very high, estimated statistically is responsible for such risks. A mathematical model is presented that describes both: the growth due to lower or over irradiated doses or the post therapy relapse of human cancer, and the shrinkage due to either of over irradiated doses, or appropriate irradiated doses. Simulations of the presented model showed that the initial tumor energy, administered dose energy, and their subsequent summation of tumor regrowth energy are always balanced with summation of Whole Body Cell Energy Burden during all treatment phases. Tumor regrows if its energy is higher than that of the dose, or if the increase of dose energy from that of the tumor is less than the one required to complete its shrinkage path. Patient-specific approaches that account for variations in tumor energies should enable more accurate dose estimates and, consequently, better protection against either lower or over irradiation that could lead to tumor regrowth and increase risks of second cancer.

Short duration treatment in genotype 1 chronic hepatitis C patients with rapid virologic response to pegylated interferon plus ribavirin

Background In patients with chronic hepatitis C, rapid HCV-RNA clearance under treatment might allow shorter treatment duration without modifying the sustained virological response (SVR) rate. This study evaluated the impact of rapid virological response (RVR) in HCV genotype 1b infection management. Methods In an open-label trial, 180 patients received standard doses of peginterferon alfa-2a plus ribavirin. Those with undetectable serum HCV-RNA at week 6 (RVR) received 24-week short-course treatment; patients with undetectable HCV-RNA at week 12 (early responders [ER]) received 48-week “standard of care” treatment; patients with positive HCV-RNA at week 12 (non-responders [NR]) stopped the treatment. Study end-point was to determine SVR rate at week 24. Results The following responses were observed: 24% RVR, 44% ER, 32% NR. Among RVR subjects, HCV-RNA baseline levels and age were significantly lower ( P = 0.038 and 0.035 respectively) than in non-RVR patients. At follow-up, 91% of RVR and 33% of ER patients achieved SVR. Among those with RVR, patients experiencing post-therapy relapse were older than those who achieved a SVR ( P = 0.028). Conclusions Chronic HCV-1b patients, achieving RVR with a 24-week treatment regimen, attained excellent SVR rates. In a cost-effective therapeutic approach, all HCV-1b patients eligible for therapy may have a short duration therapy on the basis of RVR.