Apoptosis In Neoplastic Cells Research Articles

Recent histone deacetylase inhibitors in cancer therapy.

Cancer metastasis increases the complexity of the disease and escalates patient mortality. Traditional chemotherapy has been associated with low efficacy and marked side effects. Studies pivot toward histone deacetylase (HDAC) enzymes andinhibitors because they are critical for chromatin structure, gene regulation, and cellular activities that are linked to metastasis and cancer progression. HDAC inhibitors (HDACi) can alter gene expression patterns and can lead to cell-cycle arrest and apoptosis in neoplastic cells. Several HDACi drugs like vorinostat, romidepsin, panobinostat, and belinostat are approved by the Food and Drug Administration. China and Japan have approved the use of tucidinostat, a new subtype-selective HDACi that inhibits class 1 HDAC1, HDAC2, HDAC3, as well as class 2b HDAC10. These drugs have shown promising results in the treatment of multiple carcinoma including cervical cancer, T-cell lymphoma, brain cancer, and breast cancer. This review highlights the HDACi classes, the mechanism of action of these inhibitors, their preclinical and clinical efficacy, and the latest clinical trials and patents used in cancer therapeutics. Overall, this review focuses on patents and clinical trials data from 2019 onward to give a better viewpoint on current trends in HDACis as chemotherapy agents.

Propofol elicits apoptosis and attenuates cell growth in esophageal cancer cell lines.

Propofol is a pharmaceutical agent commonly used as an intravenous anesthetic in surgical treatments and a sedative in intensive care. However, it is largely unknown how exposure to propofol affects the proliferation, invasion, and apoptosis of neoplastic cells in esophageal cancer. In this study, we sought to elucidate the impact of propofol exposure on the growth properties of human esophageal cancer cell lines in vitro. We treated two human esophageal cancer cell lines, KYSE30 and KYSE960, with up to 10 µg/mL of propofol for 12-36 h. The treated cells were then analyzed by cell proliferation assay, Matrigel invasion assay, quantification of caspase-3/7 and -9 activities, and cell staining with Annexin V and 7-aminoactinomycin D to detect early apoptosis and cell death, respectively, via flow cytometry. We found that 3-5 µg/mL propofol reduced the growth and Matrigel invasion of both cell lines in a dose-dependent manner. Executioner caspase-3/7, but not caspase-9 involved in intrinsic apoptosis pathway, was activated by cell exposure to 3-5 µg/mL propofol. In addition, 3-5 µg/mL propofol augmented early apoptosis in both cell lines and increased cell death in the KYSE30 cell line. In summary, exposure to propofol, at concentrations up to 5 µg/mL, led to the reduction of cell growth and Matrigel invasion, as well as the augmentation of apoptosis in esophageal cancer cell lines. These data will help define a methodology to safely utilize propofol, a common general anesthetic and sedative, with esophageal cancer patients.

Abstract B020: Biomarkers of IDO1 inhibition in patients with locally advanced rectal cancer treated with radiation and chemotherapy

Abstract Background: To date, immunotherapy has not advanced the initial care of locally advanced rectal cancer (LARC) where a combination of radiation and chemotherapy remains the standard of care. In evaluation of other targets, we identified that the immune-oncology target indoleamine 2,3 dioxygenase-1 (IDO1) is universally overexpressed in rectal cancers after radiation therapy (RT) regardless of MSI status. IDO1 initiates tryptophan metabolism along the kynurenine pathway (KP) and activation of the KP exerts immune-suppressive effects in the tumor microenvironment. Our preclinical data identified that epacadostat, a potent inhibitor of IDO1, reduced CRC growth in vitro and in vivo. Furthermore, we found that epacadostat augmented the anti-tumor effects of RT by directly increasing apoptosis in neoplastic cells as well as by relieving RT induced immune-suppression. Based on these findings, we initiated a Phase I study in patients with LARC where epacadostat was used in combination with short-course RT (5 Gy x 5) and chemotherapy. This study is ongoing with the primary endpoint of determining the recommended phase II dose (RP2D) of epacadostat in this population. The study is a dose escalating design including 300, 400, and 600 mg BID. Aim: To present preliminary results from the corollary studies on sequentially acquired biospecimens. Methods: We collected tumor tissue and blood pre-treatment, post-radiation/pre-chemo, sequentially throughout chemo, and at treatment completion or surgery. Analyses include global metabolomics, focused evaluation of tryptophan pathway metabolites, single cell RNAseq, flow cytometry, ctDNA and tissue analysis for IDO1 and other immune checkpoint targets. We also established tumor-derived organoids/tumoroids and have established a “tumor-on-a-chip” model to conduct ex vivo tumor modeling. Results: Preliminary studies confirm an elevation of kynurenine to tryptophan ratio after SCRT which is reversed by epacadostat. Changes in the global metabolomics profiles and immune profiles shift from baseline through the stages of SCRT and chemotherapy revealing consistent patterns. Changes in ctDNA were assessed using the CAPP-Seq based AVENIO platform providing a personalized oncogenomic analysis and demonstrated of rapid clearance from post-SCRT which persisted throughout chemotherapy. Studies with tumoroids and the analysis of scRNAseq data are ongoing and will be presented. Conclusions: Preliminary analysis of biospecimens from this actively accruing study of epacadostat plus RT and chemotherapy reveals distinct changes in metabolomic, immunologic, and genomic profiling across the stages of treatment. These results have the potential to inform a precision design for the Phase II study and will also inform the most promising disease and treatment related biomarkers. Citation Format: Matthew A. Ciorba, Katrina Pedersen, Lauren Henke, Aadel Chaudhuri, Matthew Mutch, Hyun Kim, Haeseong Park. Biomarkers of IDO1 inhibition in patients with locally advanced rectal cancer treated with radiation and chemotherapy [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B020.

Investigating Landfill Leachate Influence on Soil Microbial Biodiversity and Its Cytotoxicity

The most important problem related to the collection of waste in landfills is the presence of harmful substances contained in leachate resulting from the flow of atmospheric precipitation through the deposited waste, which may end up in the soil. As a result, the soil’s chemical degradation occurs, which may result in a change in the structure and activity of microorganisms. In addition, leachate can end up in groundwater that may be hazardous to human health. The aim of the study was to determine the effect of landfill leachate on selected physicochemical, biological and biochemical properties of soils. The cytotoxicity of soil filtrates (SF) in human cell lines and their effect on apoptosis were also analyzed. In the pot experiment, leachate was added to the soil in two doses: 50LL (50% of the N dose) and 100LL (100% of the N dose). Selected physicochemical properties, activity of enzymes, diversity of bacteria and fungi by the T-RFLP approach were determined in soil samples. As a result of the analyses, it was found that LL causes an increase in the activity of dehydrogenases and a decrease in β-glucosidase. In the soils treated with LL, bacterial T-RF (144) bp, (179) bp and (413) bp were specific, while fungal were T-RF (390) bp, (441) bp and (595) bp. Potentially cytotoxic effects of SF and reduction in apoptosis in neoplastic cells were found in in vitro tests. LL and SF are complex matrices with a variable composition, which means that their impact on the soil environment must be constantly monitored.

Vitamin D in Cancer Prevention: Gaps in Current Knowledge and Room for Hope.

Intensive epigenome and transcriptome analyses have unveiled numerous biological mechanisms, including the regulation of cell differentiation, proliferation, and induced apoptosis in neoplastic cells, as well as the modulation of the antineoplastic action of the immune system, which plausibly explains the observed population-based relationship between low vitamin D status and increased cancer risk. However, large randomized clinical trials involving cholecalciferol supplementation have so far failed to show the potential of such interventions in cancer prevention. In this article, we attempt to reconcile the supposed contradiction of these findings by undertaking a thorough review of the literature, including an assessment of the limitations in the design, conduct, and analysis of the studies conducted thus far. We examine the long-standing dilemma of whether the beneficial effects of vitamin D levels increase significantly above a critical threshold or if the conjecture is valid that an increase in available cholecalciferol translates directly into an increase in calcitriol activity. In addition, we try to shed light on the high interindividual epigenetic and transcriptomic variability in response to cholecalciferol supplementation. Moreover, we critically review the standards of interpretation of the available study results and propose criteria that could allow us to reach sound conclusions in this field. Finally, we advocate for options tailored to individual vitamin D needs, combined with a comprehensive intervention that favors prevention through a healthy environment and responsible health behaviors.

Anti-Inflammatory Nanocarriers Based on SWCNTs and Bioactive Molecules of Oregano: An In Silico Study

We studied two main bioactive molecules of oregano, carvacrol and thymol, in the present work. These bioactive conformers are linked to single wall carbon nanotubes (SWCNT) and so-called functionalized SWCNT (f-SWCNT) to find their application as anti-inflammatory drugs. We use the multiscale methods and the density functional theory (DFT) of formalism to achieve this aim. We have proposed two nanocarriers based on a finite size model of a metallic single wall carbon nanotube linked to carvacrol and thymol (with a size around 2.74 nm): the main bioactives present in oregano. The results show that the proposed molecules, Carva-SWCNT-Gluc and Thymol-SWCNT-Gluc, can be synthesized with the exposed condensation reaction; with an exergonic and spontaneous behavior, Gibbs free energies of the reaction are −1.75 eV and −1.81 eV, respectively. The studied molecules are subjected to an electronic characterization, considering the global descriptors based on the conceptual DFT formalism. Moreover, the results show that the studied molecules can present a possible biocompatibility due to the higher polarization of the molecule and the increase in apparent solubility. Finally, the interaction between the studied nanodevices (Carva-SWCNT-Gluc and Thymol-SWCNT-Gluc) with cancer and anti-inflammatory targets shows that the hydrogen bond and electrostatic interactions play a crucial role in the ligand–target interaction. The proposed f-SWCNT presents higher potentiality as a carrier vector nanodevice since it can deliver the oregano bioactives on the studied targets, promoting the putative apoptosis of neoplastic cells and simultaneously regulating the inflammatory process.

A review of biologically active flavonoids as inducers of autophagy and apoptosis in neoplastic cells and as cytoprotective agents in non‐neoplastic cells

Phytochemicals are a diverse group of compounds found in various fruits, vegetables, nuts, and legumes. Many phytochemicals have been observed to possess health benefits. Some have been found to be chemoprotective or can act as chemotherapeutics by inducing autophagy, apoptosis, or otherwise regulating the cell cycle. Many also act as potent antioxidants. Flavonoids are a subclass of bioactive phytochemicals consisting of two phenolic benzene rings, joined together by a heterocyclic pyran or pyrone. It has been observed in multiple studies that there is a correlation between diets rich in flavonoids and a reduction in cancer levels, heart disease, neurodegenerative diseases, and other pathologies. As foods containing flavonoids are widely consumed, and their mechanisms of action are still only partially understood, this review was compiled to compare the effects and mechanisms of action of some of the most widely characterized and publicized flavonoids. The flavonoids silibinin, quercetin, isorhamnetin, luteolin, curcumin genkwanin, and acacetin, together with flavonoid extracts from papaw and Tephroseris kirilowii (Turcz) Holub, a member of the Daisy family, were found to be potent regulators of the cell cycle. The decision to overview these specific flavonoids was based on their therapeutic effects, and/or their potential effects. The sparsity of data comparing these flavonoids was also a key consideration. These flavonoids all modulated to some extent the pathways of autophagy and/or apoptosis and regulated the cell cycle, inflammation, and free radical levels. This explains why they are protective of healthy or moderately damaged cells, but toxic to neoplastic or pre-cancerous cells.

Silymarin inhibits the progression of Ehrlich solid tumor via targeting molecular pathways of cell death, proliferation, angiogenesis, and metastasis in female mice.

Plant-derived phytochemicals have been reported to exert anticancer activity. This study investigated the antitumor role of silymarin (Silybum marianum) (SMN) and its molecular targets in Ehrlich solid tumor xenografts in vivo. Female Swiss albino mice were divided into three groups (of five animals each) that were engrafted with Ehrlich tumor (ET) cells with or without SMN treatment. The 3rd groups treated with DMSO only vehicle control group. A significant reduction in animal body mass and tumor volume/weight were observed in xenografted mice treated with SMN. SMN modulated oxidative stress in tumors while enhancing the antioxidant levels in mouse serum. SMN activated both mitochondrial and death receptor-related apoptosis pathways and induced cell cycle arrest, marked by a significant downregulation of cyclin D1 in SMN-treated tumors. Significant decreases in RNA content and protein expression levels of Ki-67 and proliferating cell nuclear antigen were observed in ET cells. Additionally, SMN downregulated vascular endothelial growth factor and nuclear factor-kappa B levels indicating anti-angiogenesis activity of this agent. SMN upregulated the expression of E-cadherin in tumor tissue suggesting, that SMN has potential ability to inhibit metastasis. Tumor tissue from SMN-treated animals showed a remarkable degeneration and reduction in the neoplastic cell density. The anticancer effect was associated with apparent apoptosis in neoplastic cells with abundance of multifocal necrotic areas. SMN was found to inhibit ET growth via enhancing apoptosis, inhibition of cell division and reduction in angiogenesis in vivo. Hypothetical scheme of SMN antitumor effects (mechanism of signaling) in solid ET in vivo. SMN anticancer effect may be mediated by molecular mediators that affect proliferation, cell cycle activity, apoptotic pathways, angiogenesis, and metastasis.

Association of mutations in DNA polymerase epsilon with increased CD8+ cell infiltration and prolonged progression-free survival in patients with meningiomas.

Prior studies have demonstrated a relationship between underlying tumor genetics and lymphocyte infiltration in meningiomas. In this study, the authors aimed to further characterize the relationship between meningioma genomics, CD4+ and CD8+ T-cell infiltration, and oncological outcomes of meningiomas. Understanding specific characteristics of the inflammatory infiltration could have implications for treatment and prognostication. Immunohistochemically stained meningioma slides were reviewed to assess the CD4+ and CD8+ cell infiltration burden. The relationship between immune cell infiltration and tumor genomics was then assessed using an adjusted ANOVA model. For a specific gene identified by the ANOVA, the relationship between that mutation and tumor recurrence was assessed using Cox regression. In immunohistochemically stained samples from a subcohort of 25 patients, the mean number of CD4+ cells was 42.2/400× field and the mean number of CD8+ cells was 69.8/400× field. Elevated CD8+ cell infiltration was found to be associated with the presence of a mutation in the gene encoding for DNA polymerase epsilon, POLE (51.6 cells/hpf in wild-type tumors vs 95.9 cells/hpf in mutant tumors; p = 0.0199). In a retrospective cohort of 173 patients, the presence of any mutation in POLE was found to be associated with a 46% reduction in hazard of progression (HR 0.54, 95% CI 0.311-0.952; p = 0.033). The most frequent mutation was a near-C-terminal nonsense mutation. A potential association was found between mutant POLE and both an increase in CD8+ cell infiltration and progression-free survival. The predominant mutation was found outside of the known exonuclease hot spot; however, it was still associated with a slight increase in mutational burden, CD8+ cell infiltration, and progression-free survival. Alterations in gene expression, resulting from alterations in POLE, may yield an increased presentation of neoantigens, and, thus, greater CD8+ cell-mediated apoptosis of neoplastic cells. These findings have suggested the utility of checkpoint inhibitors in the treatment of POLE-mutant meningiomas.

Effect of Betulinic acid Extraction from Guava (Psidium guajava Linn.) Leaves Against Human Cholangiocarcinoma Cells.

Background:Betulinic acid (BA), a pentacyclic triterpene glycoside extract from guava (Psidium guajava Linn.) leaves, displays a variety of biological activities which exhibit cancer therapeutic properties associated with cancer growth inhibition in various kinds of human cancer cells including brain, breast, colorectal, cervical, lung and prostate gland. However, the effects on human cholangiocarcinoma cells have not previously been reported. Current study, we evaluated the activity of BA against human cholangiocarcinoma (HuCCA) cells. Methods:The cytotoxicity analysis was measured by using MTT assay on HuCCA and BHK-21 cells. Analysis of apoptosis was evaluated by using staining with Hoechst 33342 and quantitative real-time PCR. Results:The BA (50-800 µg/mL) significantly reduced the viability of HuCCA cells in a dose-dependent action with 50% inhibitory concentration (IC50) of 92.45 µg/mL at 24 h. It also induced apoptosis signaling pathway, such as nuclear chromatin condensation and fragmentation. Quantitative real-time PCR analysis demonstrated that BA increased p53, Bax and caspase-3 expression whilst it decreased Bcl-2 expression in the HuCCA cells in a dose dependent manner. Conclusion:BA can inhibit the HuCCA cell viability and induce apoptosis of neoplastic cells. This study indicates that BA has effective treatment for cholangiocarcinoma in vitro. Consequently, BA may be used as a novel therapeutic agent for the treatment of cholangiocarcinoma in the future.

The Potential Applications of Stem Cells for Cancer Treatment.

Scientists encounter many obstacles in traditional cancer therapies, including the side effects on the healthy cells, drug resistance, tumor relapse, the short half-life of employed drugs in the blood circulation, and the improper delivery of drugs toward the tumor site. The unique traits of stem cells (SCs) such as self-renewal, differentiation, tumor tropism, the release of bioactive molecules, and immunosuppression have opened a new window for utilizing SCs as a novel tool in cancer treatment. In this regard, engineered SCs can secrete anti-cancer proteins or express enzymes used in suicide gene therapy which locally induce apoptosis in neoplastic cells via the bystander effect. These cells also stand as proper candidates to serve as careers for drug-loaded nanoparticles or to play suitable hosts for oncolytic viruses. Moreover, they harbor great potential to be employed in immunotherapy and combination therapy. However, tactful strategies should be devised to allow easier transplantation and protection of SCs from in vivo immune responses. In spite of the great hope concerning SCs application in cancer therapy, there are shortcomings and challenges to be addressed. This review tends to elaborate on recent advances on the various applications of SCs in cancer therapy and existing challenges in this regard.

Immunoexpression of REGγ and apoptosis-related proteins in oral tongue squamous cell carcinoma

The aim of this study was to investigate the immunohistochemical expression of REGγ, p53, MDM-2, Bcl-2, and Bax in oral tongue squamous cell carcinoma (OTSCC), and to correlate the findings with clinicopathological parameters. Fifty-eight OTSCC cases were selected for the study. The percentages of nuclear (REGγ, p53, and MDM-2) and cytoplasmic (Bcl-2 and Bax) staining in epithelial cells were determined and correlated with clinicopathological parameters (regional lymph node metastasis, clinical stage, clinical outcome, and histopathological grade of malignancy). Expression of REGγ was observed in all cases studied. Significantly lower percentages were observed in tumours with lymph node metastasis (P = 0.036) and in high-grade tumours (P = 0.013). No significant differences in p53, MDM-2, or Bax expression were observed according to the clinicopathological parameters. Lower percentages of Bcl-2 staining were found in high-grade OTSCC (P = 0.040) and in cases of disease-related death (P = 0.032). The expression of REGγ showed a weak positive correlation with the expression of MDM-2 (P = 0.001) and Bcl-2 (P = 0.014). The results of this study suggest that lower expression of REGγ may contribute to the progression of OTSCC. The role of REGγ in the development of OTSCC does not appear to be primarily related to the modulation of apoptosis in neoplastic cells.

Final Results of the Fibromet Trial: An Open Label Phase II Study to Evaluate Metformin Effects on Bone Marrow Fibrosis and Disease Progression in Primary Myelofibrosis Patients

▪Background: Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm characterized by myeloid expansion associated with elevation of cytokines involved in fibrosis, angiogenesis, and osteosclerosis, leading to progressive fibrous connective tissue deposition in the bone marrow (BM) and BM failure. Although JAK2, CALR and MPL mutations are frequently seen in PMF, patients' molecular heterogeneity and the lack of an expressive clinical and laboratory response following JAK1/2 inhibitors suggest that other factors, such as unknown protein interactions, additional mutations or epigenetic mechanisms may be involved in the progression of PMF. Metformin (MTF) is an anti-diabetic drug, which has been described to possess anti-cancerous properties through the modulation of the AMPK/TORC1 pathway, thereby causing apoptosis in neoplastic cells. Previous reports demonstrated that MTF significantly reduced Ba/F3 JAK2V617F tumor burden and splenomegaly in Jak2V617F knock-in-induced MPN mice. In this context, our goal was to evaluate the effects of MTF treatment in PMF patients.Aims: To report final results of an open label phase II trial (FIBROMET), which evaluated outcomes of PMF patients after 24mo on MTF treatment.Methods: PMF non-diabetic adults were eligible. Patients received MTF in increasing doses until a maximum of 2500mg PO daily, according to tolerance. Primary endpoint was BM fibrosis reversion. Secondary endpoints included reduction of inflammation and downregulation of the JAK-STAT pathway. Samples were collected at the time points: screening (0), 3mo, 6mo, 12mo, 18mo and 24mo. The extent of collagen deposits in BM biopsies was semi-quantitatively assessed with Masson's trichrome stainings, following the recommendations of the European Consensus on grading of BM fibrosis (grades 0, 1, 2 or 3). The levels of CXCL4, sIL-2Ra, IP-10, VEFG-A, MIG, MCP-1, MIP-1b, FGF-2, IL-1RA, IL-5, IL-6, IL-8, IL-15, IL-18, TNFa and TGFb1 were analyzed in BM samples using multiplex assay. Phosphorylation status of intracellular proteins STAT3 and STAT5 was analyzed by flow cytometry and the percentage of cells was recorded using FlowJo software. This trial was approved by the Institutional and National Review Board; written informed consent was obtained from all subjects. REBEC registry number: RBR-52ty66.Results: 11 patients (aged 40-84y) were included between Aug/2018- Feb/2019. Two subjects had early treatment discontinuation due to non-related causes. One patient had disease progression after 12mo of treatment and was submitted to BM transplantation. The median exposure to MTF was 21 mo (3-24) and the median dose was 2500mg/day (1500-2500mg). The most frequent adverse event was diarrhea. No life threatening event occurred. BM collagen deposits were independently evaluated by two hematopathologists with an overall agreement of 80.64% (grade 0: 100%, grade 1: 62.5%, grade 2: 60.0%, grade 3: 90.0%); discordant cases were submitted to joint review. BM collagen deposits did not change when different time points were compared. After 24mo of MTF treatment, a significant 35.4% reduction in IL-5 levels was observed (p=0.03); a 36% reduction in MCP-1 levels was also observed, however this finding was not statistically significant (p=0.06). Flow cytometry analysis demonstrated a STAT3 phosphorylation decrease when comparing screening samples versus 6, 12, 18 and 24 mo of MTF use (all p<0.05), as well as a STAT5 phosphorylation decrease when comparing screening samples versus 6 and 12 mo (all p<0.05). Mean fluorescence intensity for pSTAT3 was: screening 12.01±2.58, 3mo 7.24±1.19, 6mo 4.40±0.24, 12mo 6.31±0.47, 18 mo 6.89±1.01, 24mo 5.71±1.18; and for pSTAT5: screening 15.32±3.59, 3mo 11.50±3.74, 6mo 4.67±0.45, 12mo 6.25±0.58, 18mo 6.24±0.81, 24mo 4.98±0.72.Conclusions: Final results of the FIBROMET trial demonstrated that, in our study population, metformin was not capable of reversing established bone marrow fibrosis in PMF patients. However, a significant downregulation of the JAK-STAT pathway and reduction of cytokine secretion was observed. Finally, metformin showed to be a safe and well-tolerated drug. Phase III studies are required to confirm our results and to evaluate whether MTF treatment in early PMF phases could delay the progression of BM fibrosis through the downregulation of the JAK-STAT pathway. DisclosuresPagnano: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pintpharma: Other: Lecture; EMS: Other: Lecture; Jansenn: Other: Lecture. OffLabel Disclosure:Metformin for Primary Myelofibrosis treatment.

Hemopoiesis and Immune Cell Perturbations during Venetoclax Plus Azacytidine Treatment in Acute Myeloid Leukemia

Treatment of acute myeloid leukemia (AML) in elderly is still challenging. Indeed, high-dose chemotherapy followed by hematopoietic stem cell transplantation with myeloablative regimens is not always feasible because patients are often unfit and have several comorbidities; however, they frequently show multiple negative prognostic factors and have a worse overall survival compared to younger adults. Venetoclax, the first-in-class Bcl-2 antagonist and first approved for treatment of chronic lymphocytic leukemia, inhibits the anti-apoptotic functions of Bcl-2 inducing apoptosis and tumor growth arrest. Venetoclax is also used in combination with azacytidine, or decitabine, or low-dose cytarabine for treatment of elderly newly diagnosed AML. However, several mechanisms of resistance have already been described, such as increased expression of other anti-apoptotic proteins by the leukemic clone. In this case series, we investigated hematopoiesis and immune cell perturbations during venetoclax plus azacytidine treatment in elderly AML patients.A total of six AML patients (M/F, 2/4; median age, 71 years old; range, 63-79 years) were retrospectively evaluated at the Hematology and Transplant Center, University Hospital “San Giovanni di Dio e Ruggi d'Aragona”, Salerno, Italy. Patients received a diagnosis of AML based on the 2016 World Health Organization (WHO) criteria and chemotherapy with azacytidine 75 mg/m 2 daily for 7 days per cycle and venetoclax 70 mg/daily. Two patients were NPM1 mutated (one of them also had mutated IDH1, VAF 27.2%), while all subjects had FLT3 wild type. Based on the 2017 European LeukemiaNet risk classification, two patients had favorable risk and four intermediate. Median follow-up was 10.1 months (range, 4.9-16.6 months), and all patients were in partial or complete remission at the time of writing. Flow cytometry immunophenotype, complete blood counts (CBCs), and WT1 expression levels were performed at diagnosis and after every cycle of therapy as per our institutional guidelines.In our case series, leukemic cells were already decreased after the first cycle of therapy (blasts by flow cytometry + SD, 54.7+39.9% vs 4.2+5.4%, diagnosis vs post I cycle; P = 0.0671; paired t-test performed), while normal granulocytes detected by flow cytometry recovered only after the third cycle of therapy (20.7+23.7% vs 53+6.6%, diagnosis vs post III cycle; P = 0.1396; uncorrected Fisher's mixed model performed). Treated patients also displayed a contextual decreased in WT1 expression levels (normalized WT1 copy number + SD, 1810+2723 copies vs 201+132.9 copies, diagnosis vs post I cycle; P = 0.2660; paired t-test performed). Platelet count tended to increase after the first cycle (P = 0.0680); however, at the end of the second cycle, half of patients were again thrombocytopenic (platelets < 100 x 10 3/µL). Interestingly, percentage of lymphocytes detected by flow cytometry were significantly increased after the second cycle of azacytidine plus venetoclax compared to baseline and after the first cycle of therapy (mean+SD, 13.5+13.3% vs 48+8.7%, diagnosis vs post II cycle; P = 0.0167; and vs 28+11.3%, vs post III cycle; P = 0.0480), likely because an increase in Natural Killer (NK) cell frequency peaking after the second cycle (mean+SD, 19.4+4.4% vs 32.5+15.1%, diagnosis vs post II cycle; P = 0.1383). Moreover, five out of six patients displayed expansion of plasma cells detected by flow cytometry in the bone marrow after the first cycle: in particular, one case patient had expansion of aberrant CD45-/dimCD38++CD138++CD56+CD19- plasma cells, while one subject showed only a transient appearance of clonal plasma cells after the second cycle. No differences in bone marrow monocyte frequencies were described during treatment.Our preliminary results added evidence to efficacy and safety of the combination of venetoclax and azacytidine in treatment of elderly AML in a real-world setting. These drugs might synergistically function on hematopoiesis by inducing apoptosis of neoplastic cells while favoring differentiation of other lineages, as suggested by the expansion of plasma cells, or triggering NK-mediated immunosurveillance. However, prognostic and clinical significance of plasma cell and NK cell expansion in the setting of AML treatment needs to be further explored in larger prospective cohorts. DisclosuresNo relevant conflicts of interest to declare.

Preparation and characterization of gadolinium-based thermosensitive liposomes: A potential nanosystem for selective drug delivery to cancer cells

Gadodiamide (Gd-DTPA-BMA) is a gadolinium-based complex, used as a contrast agent in magnetic resonance imaging procedures. Recent studies revealed the capacity of this complex of inducing apoptosis in neoplastic cells. However, a great challenge for its use as an anticancer drug is low cellular internalization and drug delivery systems such as thermosensitive liposomes can be a strategy to overcome these limitations and increase anti-tumor efficacy. In this context, this study aimed to develop, characterize, and assess the cytotoxic activity and selectivity index of thermosensitive liposomes containing Gd-DTPA-BMA. Formulations were prepared by the lipid film hydration method and their physicochemical, morphological, and thermal properties were evaluated. Cell viability was performed 4T1/MDA-MB-231 tumor cells and WI-26 VA4 normal cells. Transmission electron microscopy analyses showed high electron density in the inner core of Gd-DTPA-BMA-loaded liposomes that can be attributed to the Gd-DTPA-BMA, since Gd-loaded nanosystems present low light transmittance and high electron density by this technique. The dynamic light scattering, differential scanning calorimetry, small-angle X-ray scattering, and in vitro release results confirm that the lipid composition was suitable since Tc (temperature of the transition Lβ → Lα) values are in accordance with those reported for thermosensitive liposomes used in the treatment of cancer. The cytotoxic studies against breast cancer cell lines demonstrated that the Gd-DTPA-BMA-loaded liposomes have higher cytotoxicity than free Gd-DTPA-BMA. Moreover, these liposomes presented minimal toxicity in a normal cell line when compared to the free Gd-DTPA-BMA. Therefore, the results of this study suggest that thermosensitive liposomes containing Gd-DTPA-BMA might be a new promising nanocarrier system for breast cancer treatment.

Proliferation to Apoptosis Tumor Cell Ratio as a Biomarker to Improve Clinical Management of Pre-Malignant and Symptomatic Plasma Cell Neoplasms.

Proliferation and apoptosis of neoplastic cells are prognostic biomarkers in plasma cell neoplasms (PCNs). The prognostic capacity of proliferation to apoptosis ratio (Ratio-PA) in the era of immunomodulatory treatments is re-evaluated in 316 gammopathy of undetermined significance (MGUS), 57 smoldering multiple myeloma (SMM), and 266 multiple myeloma (MM) patients. Ratio-PA of 0.77 ± 0.12, 1.94 ± 0.52, and 11.2 ± 0.7 (p < 0.0001) were observed in MGUS, SMM, and MM patients. Ten-year overall survival (10y-OS) rates for patients with low/high Ratio-PA were 93.5%/77.3% p < 0.0001) for MGUS, 82.5%/64.7% (p < 0.05) for SMM, and 62.3%/47.0% (p < 0.05) for MM. For patients with low, intermediate, and high risk, 10y-OS for low/high Ratio-PA were 95.5%/72.9% (p < 0.0001), 74.2%/50.4% (p < 0.0001), and 35.3%/20.0% (p = 0.836), respectively. Ratio-PA was an independent prognostic factor for OS (HR = 2.119, p < 0.0001, Harrell-C-statistic = 0.7440 ± 0.0194) when co-analyzed with sex, age, and standard risk. In patients with Ratio-PAhigh, only first-line therapy with VRd/VTd, but not PAD/VCD, coupled with ASCT was associated with high 10y-OS (82.7%). Tumor cell Ratio-PA estimated at diagnosis offers a prognostic biomarker that complements standard risk stratification and helps to guide the clinical management of pre-malignant and symptomatic PCNs. Every effort should be made to provide first-line therapies including VTd or VRd associated with ASCT to patients with Ratio-PAhigh at higher risk of progression and death.

Genomic Landscape of Hodgkin Lymphoma.

Simple SummaryHodgkin lymphoma (HL) is composed of many reactive and only a few cancer cells, so-called Hodgkin and Reed-Sternberg (HRS) or lymphocyte predominant (LP) cells. Due to the scarcity of these cells, it was difficult to perform high-throughput molecular investigations on them for a long time. With the help of recently developed methods, it is now possible to analyze their genomes. This review summarizes the genetic alterations found in HRS and LP cells that impact immune evasion, proliferation and circumvention of programmed cell death in HL. Understanding these underlying molecular mechanisms is essential, as they may be of prognostic and predictive value and help to improve the therapy especially for patients with recurrent or treatment-resistant disease.Background: Hodgkin lymphoma (HL) is predominantly composed of reactive, non-neoplastic cells surrounding scarcely distributed tumor cells, that is, so-called Hodgkin and Reed-Sternberg (HRS) or lymphocyte predominant (LP) cells. This scarcity impeded the analysis of the tumor cell genomes for a long time, but recently developed methods (especially laser capture microdissection, flow cytometry/fluorescence-activated cell sorting) facilitated molecular investigation, elucidating the pathophysiological principles of “Hodgkin lymphomagenesis”. Methods: We reviewed the relevant literature of the last three decades focusing on the genomic landscape of classic and nodular lymphocyte predominant HL (NLPHL) and summarized molecular cornerstones. Results: Firstly, the malignant cells of HL evade the immune system by altered expression of PDL1/2, B2M and MHC class I and II due to various genetic alterations. Secondly, tumor growth is promoted by permanently activated JAK/STAT signaling due to pervasive mutations of multiple genes involved in the pathway. Thirdly, apoptosis of neoplastic cells is prevented by alterations of NF-κB compounds and the PI3K/AKT/mTOR axis. Additionally, Epstein-Barr virus infection can simultaneously activate JAK/STAT and NF-κB, similarly leading to enhanced survival and evasion of apoptosis. Finally, epigenetic phenomena such as promoter hypermethylation lead to the downregulation of B-lineage-specific, tumor-suppressor and immune regulation genes. Conclusion: The blueprint of HL genomics has been laid, paving the way for future investigations into its complex pathophysiology.

Inhibition of Wnt/Beta-Catenin Pathway Impairs Natural Killer (NK) Cells Induced Apoptosis of Myeloid Leukemia Cells

Introduction:NK cells play an important role in the immune response to cancer due to their ability to induce T-cell independent apoptosis of neoplastic cells. NK cells recognize tumor cells through the interaction of several distinct cell surface receptors, with the net result of inducing NK cell activation and cytotoxic attack on tumor cells. It is hypothesized that a failure of NK surveillance is a contributing factor in the development of acute myeloid leukemia (AML). The discovery of pathways that regulate AML cell apoptosis in response to NK cells activation may represent a potential therapeutic target. It is known that the Wnt/beta-catenin pathway regulates cellular proliferation, differentiation and resistance to apoptosis in AML cells. However, its contribution to the resistance of AML cells in response to NK cell-induced apoptosis resistance is currently unknown. Objective:The objective of the current study is to determine if inhibition of the Wnt/beta catenin pathway impacts NK cell-induced apoptosis of AML cells. Methods:HL-60 leukemia cells were treated with different concentrations of the Wnt/beta-catenin pathway inhibitor XAV-939 (tankyrase inhibitor) to determine the lowest dose capable of inhibiting the Wnt/beta-catenin pathway without inducing cell apoptosis. Inhibition of the pathway was confirmed by evaluating beta-catenin protein by intracellular flow cytometry and real-time PCR for downstream factors MYC and TCF4. Next, differentiation of HL-60 cells into mature granulocytes was evaluated by analyzing expression of CD11b by flow cytometry after stimulation with dimethyl sulfoxide (DMSO), as another marker of Wnt/Beta-catenin pathway inhibition. After confirming pathway inhibition, HL-60 cells were incubated with healthy donor derived NK cells in a 2:1 ratio for 24 hours in the presence or absence of XAV-939. Apoptosis was evaluated by annexin staining. Expression of NK cell inhibitory proteins were determined by flow cytometry. A panel of 84 genes for hematopoietic transcription factors was evaluated by PCR array (RT2 Profiler PCR Array Gene Expression - Qiagen) in HL-60 cells after exposure to XAV-939 for 2 and 6 hours. Expression of apoptosis related genes in HL-60 cells after exposure to XAV-939 was determined by real-time PCR. Results:The dose of 1mM XAV-939 was selected to evaluate is effect in the HL-60 cell line. Suppression of pathway signaling was confirmed by a decrease in expression of Beta-catenin,MYC,TCF4and blockade of HL-60 differentiation into neutrophils. After coincubation with NK cells, there was a decrease in HL-60 cell death with concomitant exposure to XAV-939 (70% [untreated] vs. 30% [XAV-939]; p&amp;lt;0.05) (figure 1). There was no change in NK cell surface receptors or their putative ligands in HL-60 cells as evaluated by flow cytometry. Analysis of expression of hematopoietic transcription factor in HL-60 cells showed a downregulation of several transcription factors after exposure to XAV-939, includingJUNDandTCF7L2. Since there was a decrease in apoptotic cell death, we next evaluated expression of genes related to apoptotic pathways and could demonstrate a decrease in expression ofFASandTNFR1after exposure to XAV-939 (p&amp;lt;0.05) (figure 2A and 2B). Conclusions:These results suggest that inhibition of the Wnt/beta-catenin pathway decreases apoptosis of myeloid leukemia cells mediated by NK cells, and this effect may be secondary to a decreased expression ofFASandTNFR1in the leukemic cells. Figure Disclosures Santos: Novartis:Other: Speaker fee;Bristol-Myers Squibb:Other: Speaker fee.

Restoring Apoptosis with BH3 Mimetics in Mature B-Cell Malignancies.

Apoptosis is a highly conserved mechanism enabling the removal of unwanted cells. Mitochondrial apoptosis is governed by the B-cell lymphoma (BCL-2) family, including anti-apoptotic and pro-apoptotic proteins. Apoptosis evasion by dysregulation of anti-apoptotic BCL-2 members (BCL-2, MCL-1, BCL-XL) is a common hallmark in cancers. To divert this dysregulation into vulnerability, researchers have developed BH3 mimetics, which are small molecules that restore effective apoptosis in neoplastic cells by interfering with anti-apoptotic proteins. Among them, venetoclax is a potent and selective BCL-2 inhibitor, which has demonstrated the strongest clinical activity in mature B-cell malignancies, including chronic lymphoid leukemia, mantle-cell lymphoma, and multiple myeloma. Nevertheless, mechanisms of primary and acquired resistance have been recently described and several features such as cytogenetic abnormalities, BCL-2 family expression, and ex vivo drug testing have to be considered for predicting sensitivity to BH3 mimetics and helping in the identification of patients able to respond. The medical need to overcome resistance to BH3 mimetics supports the evaluation of innovative combination strategies. Novel agents including MCL-1 targeting BH3 mimetics are currently evaluated and may represent new therapeutic options in the field. The present review summarizes the current knowledge regarding venetoclax and other BH3 mimetics for the treatment of mature B-cell malignancies.

Abstract 4753: Attenuating colorectal cancer cell growth and enhancing immunogenicity through rational combination of MEK and HDAC inhibition

Abstract Background: Studies have shown that MEK inhibitors (MEKi) can reduce tumorigenic growth signaling in colorectal cancer (CRC) through inhibition of the RAS pathway which is upregulated in over 40% of CRC tumors, while HDAC inhibitors (HDACi) lead to cell cycle arrest, inhibit angiogenesis, and induce apoptosis in neoplastic cells. In addition to a direct effect on neoplastic cells, MEKi have also demonstrated the potential to increase immune response to the tumor through upregulation of MHC I on tumor cells, induction of intra-tumoral T-cell infiltration, and enhancement of anti-PDL1 activity. HDAC inhibitors lead to epigenetic alterations which have similarly shown an ability to increase tumor immunogenicity, leading to the hypothesis that these agents may be combined with immunotherapy treatments for enhanced anti-tumor effect. In this study we first analyzed the effect of MEKi and HDACi combination treatment in both resistant and sensitive CRC cell lines to assess for synergy in the attenuation of cancer cell proliferation. We then demonstrated the effect of combination MEKi and HDACi therapy on tumor immunogenicity through increased expression of tumor immune markers. Methods: We assessed the efficacy of a novel MEKi agent, HL-085, in comparison with trametinib in six different CRC cell lines. Treatment with each agent was separately administered with a maximum concentration of 10 uM. Similarly, five different HDAC inhibitors were tested in each of these cell lines, with a maximum concentration of 1 uM. Optimal combination of MEK inhibitor HL085 and HDAC inhibitor class I/II (OKI-005) were then chosen based on this data. Cellular proliferation was assessed after 72 hours with Cell Titer Glo. Flow cytometry of a panel of immunogenic markers was then performed alongside a viability stain to assess for expression level changes of MCH class I in CRC cells. Results: Combinatorial effects of dual treatment with MEK inhibitor HL085 and HDAC inhibitor OKI-005 are observed in both resistant and sensitive cell lines with synergistic effect produced in the SW620 and Lovo cell lines (MEKi sensitive) as well as the GP2D (MEKi resistant) cell line. No synergistic effect was observed in the HCT15, DLD1 and LS513 cell lines. Flow cytometry showed an increase in MHC class I expression in neoplastic cells with combined MEKi and HDACi treatment compared to individual agent treatment and control, demonstrating increased immunogenicity of CRC cells with combination treatment. Conclusions: Combination treatment with a novel MEK inhibitor HL085 and the HDAC inhibitor OKI-005 demonstrates synergy in three out of six colorectal cancer cell lines. We further show increased immunogenicity of CRC cells through enhanced expression of MCH I on tumor cell surface, indicating rationale for further investigation of combination MEKi and HDACi with immunotherapy in the treatment of metastatic colorectal cancer. Citation Format: Mihailo Miljanic, Nisha Holay, Anna Capasso, Todd Triplett, Milad Soleimani, Uma Giri, Carla Van Den Berg, Gail Eckhardt. Attenuating colorectal cancer cell growth and enhancing immunogenicity through rational combination of MEK and HDAC inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4753.