Line Agents Research Articles

Survival and Outcomes after Relapse in Newly Diagnosed Acute Myeloid Leukemia (AML) Patients Treated with Venetoclax-Based Chemotherapy

Background Venetoclax (bcl-2 inhibitor) was first approved in 2018 for use in combination with hypomethylating agents (HMA) or low-dose cytarabine (LDAC) for treatment of newly diagnosed acute myeloid leukemia (AML) in older patients, or those who have comorbidities that preclude use of intensive induction chemotherapy. However, AML is also known to evolve and undergo clonal selection when treated with venetoclax, leading to treatment failure. Mechanisms for adaptive resistance include kinase activation (e.g. FLT3-ITD), clones that acquire biallelic silencing of TP53, and monocytic AML cells that upregulate MCL1 to evade BCL2 blockade. Other issues with venetoclax-based regimens include prolonged cytopenias and increased risk of infection. Our aim was to investigate survival after relapse, subsequent responses to second- line agents, and pattern of infections in patients treated with first-line venetoclax-based combination therapies. Methods This was a retrospective study of patients who received venetoclax-based regimens for untreated AML or myelodysplastic syndrome with excess blasts (MDS-EB) at LAC+USC Medical Center and Norris Cancer Center in Los Angeles, CA from January 1, 2018 to June 30, 2022. The electronic medical record was reviewed for demographics, disease characteristics, clinical history, transplant status, and survival. Results 61 patients received frontline venetoclax-based regimens for untreated AML or MDS-EB with a mean age of 65 (range 22-84); 36% were female, 44% non-Hispanic white and 31% were Hispanic/Latino. 41% had AML-MRC (myelodysplasia-related changes), 36% had De Novo AML, 16% had therapy-related AML, and 7% had MDS-EB. The majority (70%) were adverse risk group per ELN 2017 and 46% had complex cytogenetics. 58 patients had available myeloid mutation panel at time of diagnosis. The most common mutations (median allele frequencies) were: TP53=33% (0.45), ASXL1=17% (0.37), NPM1=16% (0.39), IDH2=9% (0.42), IDH1=7% (0.29), FLT3-ITD=5% (0.51). 70% received venetoclax with decitabine, 21% received venetoclax with azacitidine, and 2% received venetoclax with LDAC. Median number of cycles was 3 (1-16). 10 patients underwent allogeneic stem cell transplant. Out of 61 patients, 12 (20%) had primary refractory disease and 23 (38%) relapsed on frontline venetoclax. 49% of these relapsed/refractory patients received salvage chemotherapy. The median time to relapse and number of cycles prior to relapse was 6.3 months (1.6-17.4) and 4 (1-12) respectively. There was no correlation between cytogenetics and genetic variables and risk of relapse. Only 3 (19%) out of 16 patients and 3 (27%) out of 11 patients who received second- and third-line treatment achieved CRi respectively. Myeloid mutation panel was available for 12 patients at time of relapse. Most common mutations were FLT3-ITD/TKD (n=5), TP53 (n=3) and NPM1 (n=3). Six patients acquired new mutations at the time of relapse: FLT3-ITD (n=2), TP53 (n=1), WT1 (n=2) and ETV6 (n=1). Median time from relapse to death was 3 months (1-23). Median overall survival was 10.2 months. Median survival in responder compared to non-responders after cycle 1 and cycle 2 was 16.8 vs. 5.3 months (p <0.001) and 19.4 months vs. 7.4 months (p=0.031) respectively. Median survival in patients with relapse compared to non-relapse was 12.1 vs 6 months (p=0.187). 40 (66%) patients died; major causes of death were disease progression (50%), bleeding (10%) and infection (25%). 28% patients died in intensive care unit. 9 patients had fungal infections; most common were aspergillosis (n=5) and mucormycosis (n=2). Conclusions Venetoclax-based regimens are effective options for untreated AML in elderly patients. Response to first and second cycle of combination treatment predict survival. Although we observed clonal evolution in relapsed patients, it did not affect their survival. FLT3-ITD was a commonly acquired mutation at time of relapse, thus patients received salvage with targeted agents. Also, as patients relapse, the chance of attaining CR/CRi with subsequent lines of treatment is less than 30% and risk of invasive fungal infection was approximately 15%. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Nanoliposomal C6-Ceramide Exhibits Synergistic Anti-Cancer Activity with a Small Molecule Agonist of PP2A in Chronic Lymphocytic Leukemia

The introduction of targeted therapies, including venetoclax (inhibitor of Bcl-2) and ibrutinib (inhibitor of Bruton's tyrosine kinase), has significantly advanced treatment of non-Hodgkin's lymphoma, particularly Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL). However, patients treated with ibrutinib, venetoclax, or the combination of ibrutinib and venetoclax are likely to exhibit treatment relapse. We have previously published that microenvironmental factors can generate anti-apoptotic resistance to combinatorial ibrutinib/venetoclax in both CLL and MCL models via NF-kB-dependent upregulation of anti-apoptotic proteins (Mcl-1 and Bcl-xL) (Jayappa et al. 2017). We have recently published the discovery of phenotypically similar drug resistant CLL cells that overexpress multiple anti-apoptotic proteins in patients de novo. These cells were enriched during treatment with venetoclax, suggesting this pool of resistant persister cells could be a source of treatment failure (Jayappa et al. 2021). We have previously formulated a non-toxic C6-ceramide nanoliposome (CNL) that greatly improves bioavailability of this bioactive sphingolipid and exerts anti-cancer activity in vivo, including CLL cell line that exhibits multidrug resistance phenotype (Lindsay et al. 2013). Thus, CNL-based novel therapies could hold promise for depleting the pool of multidrug resistant cancer cells in patients. In the current study, we screened CNL activity in CLL patient PBMC samples (N=15) using a flow cytometry-based cytotoxicity assay ex vivo. Our analysis revealed that CNL induced apoptotic/non-apoptotic cell death at overly high concentrations (EC50, 31 µM), prompting us to search for potential combinatorial therapies with enhanced toxicity profiles. To address this, we performed a combination drug screen with CNL as an anchor agent in a multidrug resistant CLL cell line (MEC1, p53-/mt) using a cell survival assay. Our analysis discovered exceptional synergistic interaction between CNL and a small molecule activator of Protein Phosphatase 2A (PP2A) (SMAP, DT061) in MEC1 cells [Bliss score (actual-Bliss predicted cytotoxicity): 56% (CNL, 6 µM; DT061, 12 µM)] and this synergistic behavior was further validated in primary CLL samples (N=5) ex vivo. Analyses using patient PBMC samples (N=3) have shown that the CNL and SMAP combination triggers apoptotic cell death with cleavage of caspase9, capase3, and PARP in CLL cells, while normal T lymphocytes in these samples were relatively less affected. At the molecular level, mutually reinforcing interactions between SMAP and CNL were observed. Using mass-spectrometric profiling of sphingolipids, we discovered that PP2A activation by SMAP modulates sphingolipid metabolism, leading to enhanced accumulation of C6-ceramide as well as synthesis of endogenous, pro-apoptotic sphingolipid molecules (C14- and C16-ceramides) in cells exposed to CNL. In addition, co-treatment with SMAP and CNL induced synergistic/additive downregulation of pBAD and cMyc, suggesting cMyc and/or Bad-dependent pathways could serve as node of convergence for synergistic interaction seen in these cells. In summary, we demonstrate that the CNL-SMAP combination displays exceptional synergy in CLL cells. At the molecular level, CNL and SMAP exhibit mutually reinforcing interactions that potentially converge on cMyc and Bad pathways, leading to synergistically increased apoptotic death in these cells. We intend to further characterize the role of cMyc and/or Bad pathway in synergistic interaction and assess the in vivo safety/efficacy of CNL-SMAP combination using a CLL xenograft mouse model. Collectively, this work provides the basis for further testing of this combination in in vivo models.

Menin Inhibitor DS-1594b Drives Differentiation and Induces Synergistic Lethality in Combination with Venetoclax in AML Cells with MLL-Rearranged and NPM1 Mutation

Introduction: Mixed-lineage-leukemia (MLL) is one of the most frequently translocated genes in hematologic malignancies, which produces aggressive leukemias that are refractory to conventional treatment resulting in a 5-year survival rate of only 35%. MLL translocations lead to fusion of the N-terminal fragment of MLL with one of over sixty protein partners, producing MLL fusion proteins that enhance proliferation, block hematopoietic differentiation, and drive leukemia development. Acute myeloid Leukemia (AML) is a heterogeneous and rapidly progressing blood cancer with a poor prognosis, especially in elderly patients where it is more common. Mutations in the nucleophosmin (NPM1) gene are one of the most common causes of AML. Interestingly, the protein-protein interaction between Menin, MLL and MLL fusion proteins plays a critical role in the pathogenesis of acute myeloid leukemias, both with MLLr as well as in leukemias carrying mutations in NPM1. Inhibition of the MLL-Menin interaction with novel Menin inhibitors has shown efficacy in both AML subsets, in pre- clinical and clinical studies. However, the responses are short-lived and hence it is imperative to identify therapeutic approaches that may synergize with Menin inhibition. Our recent work showed that MLLr t(4;11) Acute Lymphoblastic leukemia () patient cells express high levels of the anti-apoptotic BCL-2 protein and are highly sensitive to treatment with the BCL-2 selective antagonist, venetoclax (Benito, J.M., et al , Cell Reports, 2015). Here, we report experiments to establish the efficacy of the novel Menin-MLL inhibitor DS-1594b which was developed by Daiichi Sankyo co., jp, in combination with the BCL-2 antagonist venetoclax (VEN) in MLLr and NPM1-mutated acute leukemias. Methods: The efficacy of DS-1594b/VEN combination in MLLr and NPM1mut AML cell lines and primary leukemia samples was evaluated by examining the cell viability in response to serial dilutions of tested compounds as single agents or in combination using Cell Titer Glo (CTG) assay at 3, 5 and 7 days of treatment. Induction of apoptosis was assessed by Annexin V/ staining. Since down-regulation of MLL fusion target genes leads to differentiation in MLL leukemic cells, we monitored cell differentiation by flow cytometry in AML cell lines (MLLr: OCI-AML2, MOLM13, MOLM14, MV411. NPM1 Mut: OCI-AML3 . Leukemia cell lines with no MLLr or NPM1 mutations: HL60, U937) and primary leukemia samples ( blood from relapsed MLLr and NPM1mut AML) using differentiation markers CD11b, CD14, CD15. MLLr cell line MOLM13 resistant to venetoclax was also included in these studies. Results: 7 days treatment with DS-1594b reduced viability in MLLr and NPM1mut cell lines at low nM concentrations but did not affect growth of negative control U937 and HL60 . DS-1594b induced differentiation in OCI-AML3 and Ven resistant cell after 5 days and in other lines after 10 days (figure 1A). DS-1594b did not induce apoptosis when used as a single agent in any AML cell line tested indicating that differentiation is a key mechanism of Menin inhibition. Five days of treatment with single agent showed a downregulation of Menin in all cell lines and downregulation of BCL2 in OCI-AML3 and OCI-AML2 in Western blot analysis. Remarkably, combination with Venetoclax induced synergistic lethality and growth inhibition in all cell lines. Most importantly, was observed in primary leukemia samples (Figure 1B), with differentiation effects after DS-1594b single agent (bottom). Conclusions: Here we show in vitro efficacy of DS-1594b, a selective small molecule menin inhibitor, as single agent or in combination with Venetoclax in MLLr and NPM1 mutated cell lines and primary leukemia samples. Preliminary data demonstrate differentiation effects of DS-1594b as single agent in both cell lines and primary AML leukemia samples. Combination effects show synthetic lethality in almost all cell lines except OCI-AML3. The combination elicited profound apoptosis in primary leukemia samples. In vivo studies using MLLr and NPM1 PDXs models are ongoing and will be presented. Overall, the preliminary findings presented here pave the way for further studies aimed at understanding mechanisms of and further development and testing of this combination in AML with MLLr and/or NMP1 mutations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical and Demographic Characteristics of Multiple Myeloma Patients at Study Enrollment: Analysis of Doriant Study (NCT:04659798)

BACKGROUND: Multiple Myeloma is a heterogenous disease characterized by subsequent relapses in which the burden of the disease and previous treatments received could limit subsequent lines of therapy, as described in treatment guidelines. The disease becomes more aggressive after each relapse and the time between relapses is shorter, which means less duration of response per late line of treatment that could lead into treatment refractoriness. Progression free survival is shorter after each treatment line and the prognosis of the patients gets worse. Increasing the DoT at relapse has a positive impact in the overall survival (OS) and in the clinical evolution of the disease. However, duration of treatment (DoT) is not reported outside clinical trials and there is a lack of information in clinical practice. There is increasing evidence to Support a treatment paradigm shift from the traditional fixed duration strategy to a continuous therapy approach to improve outcomes. The determinants of DoT and its relationship with survival in real-life practice have been little investigated, including reasons for treatment discontinuation. This research aims to investigate the relationship between DoT and outcomes in clinical practice, also analyzing the satisfaction with the treatment received from patients with MM and AL amyloidosis as an exploratory endpoint. OBJECTIVE: To investigate the relationship between DoT and response according to selected treatment strategy (fixed vs continuous) among patients diagnosed with MM and/or AL amyloidosis. We provide an update regarding baseline characteristics of the MM population enrolled in the study as per the information collected during the entry study visit. METHODS: Prospective, non-interventional, multi-centre study in real-life practice in 14 Spanish hospitals. The study enrolled subjects aged 18 or older diagnosed with MM and/or AL amyloidosis who started treatment up to 12 months before the inclusion. The study has enrolled a total of 240 patients, 207 of them with MM diagnosis. RESULTS: Of the 207 MM enrolled patients, 191 (92,3%) were eligible for this analysis. 106 (55.5%) were at 1L of treatment, 52 (27.2%) at 2L, 24 (12.6%) at 3L and 9 (4.7%) at subsequent lines. 30 patients were at maintenance setting. The median age was 74 (67,79) years, 93 (48,7%) were male and 98(51,3%) were female. 142 (75,1%) lived in urban areas, 16 (8.9%) patients lived alone, 154 (86%) with the family and 9 (5%) requires support from a caregiver. At study entry, 143 (78.1%) were independent, 25 (13.7%) grade I, 12 (6.6%) grade 2 and 3(1.6%) grade 3. 42 patients (22.3%) have primary studies, 14 (7.5%) were in active employment, 11 (5.9%) unemployed and 146 (78.5%) were retired. Physical activity was high in 10 (5.5%) patients, 102 (56.4%) moderate/low and 69 (38.1%) were inactive. Comorbidities are listed in Table 1. 44.5% of the patients have received a previous line of treatment and 60 (31.6%) has received a previous stem cell transplant (SCT) at study visit. Laboratory and cytogenetic test were available in 189 patients at study entry. 25 (13.3%) have minimal residual disease (MRD) test available with 9.6% positive and 3.7% negative. 90 (47.6%) patients performed a cytogenetic abnormalities test at current treatment initiation, 39 (43.3%) with high risk and 51 (56.7%) with standard risk. ECOG at current treatment initiation was available in 89 patients, 23 (25.8%) ECOG 0, 56 (62.9%) ECOG 1 and 7 (7.9%) ECOG 2. In 190 patients Charlson Comorbidity Index was performed at current treatment initiation and 184 patients completed the TSQM questionnaire. Prescribed regimens per line and main agent are listed in table 2. 41 (21,5%) patients received a fixed duration vs 150 (78,5%) continuous treatment strategy. CONCLUSION: Based on this preliminary analysis in the MM eligible population, the majority of patients are under a continuous treatment strategy, are in early treatment lines and the current treatment received is based on monoclonal antibodies as main agent. Further analysis will be provided to determine treatment patterns per line and duration of treatment prescribed in real-life practice. Keywords: multiple myeloma (MM); clinical characteristics, duration of treatment, fixed and continuous treatment, real-life practice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Resistance Mechanisms to Anti-Epidermal Growth Factor Receptor Therapy in RAS/RAF Wild-Type Colorectal Cancer Vary by Regimen and Line of Therapy.

Acquired resistance to anti-epidermal growth factor receptor (EGFR) inhibitor (EGFRi) therapy in colorectal cancer (CRC) has previously been explained by the model of acquiring new mutations in KRAS/NRAS/EGFR, among other MAPK-pathway members. However, this was primarily on the basis of single-agent EGFRi trials and little is known about the resistance mechanisms of EGFRi combined with effective cytotoxic chemotherapy in previously untreated patients. We analyzed paired plasma samples from patients with RAS/BRAF/EGFR wild-type metastatic CRC enrolled in three large randomized trials evaluating EGFRi in the first line in combination with chemotherapy and as a single agent in third line. The mutational signature of the alterations acquired with therapy was evaluated. CRC cell lines with resistance to cetuximab, infusional fluorouracil, leucovorin, and oxaliplatin, and SN38 were developed, and transcriptional changes profiled. Patients whose tumors were treated with and responded to EGFRi alone were more likely to develop acquired mutations (46%) compared with those treated in combination with cytotoxic chemotherapy (9%). Furthermore, contrary to the generally accepted hypothesis of the clonal evolution of acquired resistance, we demonstrate that baseline resistant subclonal mutations rarely expanded to become clonal at progression, and most remained subclonal or disappeared. Consistent with this clinical finding, preclinical models with acquired resistance to either cetuximab or chemotherapy were cross-resistant to the alternate agents, with transcriptomic profiles consistent with epithelial-to-mesenchymal transition. By contrast, commonly acquired resistance alterations in the MAPK pathway do not affect sensitivity to cytotoxic chemotherapy. These findings support a model of resistance whereby transcriptomic mechanisms of resistance predominate in the presence of active cytotoxic chemotherapy combined with EGFRi, with a greater predominance of acquired MAPK mutations after single-agent EGFRi. The proposed model has implications for prospective studies evaluating EGFRi rechallenge strategies guided by acquired MAPK mutations, and highlights the need to address transcriptional mechanisms of resistance.

Triazole-Bridged Zinc Dinuclear Complexes: Mechanochemical Synthesis, Crystal Structure, and Biological Activity.

Two zinc (Zn) complexes, [Zn2(DAT)2Cl4] (I) and [Zn2(DAT)2(NO3)4] (II), were prepared by grinding 3,5-diamino-1,2,4-triazole (C2H5N5, DAT) with Zn precursors such as ZnCl2 and Zn(NO3)2, respectively. This solid-state reaction gives the corresponding Zn complex as the sole product in over 99% yield. This mechanochemical method promotes the selective formation of Zn complexes different from those obtained using the conventional solution-based route. The crystal structures of the two complexes were analyzed by single-crystal X-ray diffraction. Complex (I) crystallizes in the monoclinic space group P21/c, whereas complex (II) crystallizes in the triclinic space group P 1̅. Each complex is characterized by the presence of a characteristic DAT-bridged dimer with one DAT ligand per Zn atom, and the DAT ligand provides a bridge between the two Zn metals. All Zn centers of (I) and (II) adopted a slightly distorted tetrahedral geometry. Both complexes contain a hexanuclear Zn2N4 ring, but their ring structures are different. Complex (I) possesses a boat geometry, while complex (II) has a nearly planar structure. The Zn-bound chlorides of complex (I) form intermolecular N-H···Cl hydrogen bonds that link neighboring molecules. In complex (II), the O atoms in the nitrate groups are hydrogen-bonded to the DAT ligand via O···H-N linkages. Both complexes exhibit blue emissions in the solid state at ambient temperature. They were evaluated as anticancer agents in HeLa, NCCIT, and MCF-7 cancer cell lines, exhibiting promising anticancer activities.

Repurposing FDA-approved drugs as inhibitors of therapy-induced invadopodia activity in glioblastoma cells

Glioblastoma (GBM) is the most prevalent primary central nervous system tumour in adults. The lethality of GBM lies in its highly invasive, infiltrative, and neurologically destructive nature resulting in treatment failure, tumour recurrence and death. Even with current standard of care treatment with surgery, radiotherapy and chemotherapy, surviving tumour cells invade throughout the brain. We have previously shown that this invasive phenotype is facilitated by actin-rich, membrane-based structures known as invadopodia. The formation and matrix degrading activity of invadopodia is enhanced in GBM cells that survive treatment. Drug repurposing provides a means of identifying new therapeutic applications for existing drugs without the need for discovery or development and the associated time for clinical implementation. We investigate several FDA-approved agents for their ability to act as both cytotoxic agents in reducing cell viability and as ‘anti-invadopodia’ agents in GBM cell lines. Based on their cytotoxicity profile, three agents were selected, bortezomib, everolimus and fludarabine, to test their effect on GBM cell invasion. All three drugs reduced radiation/temozolomide-induced invadopodia activity, in addition to reducing GBM cell viability. These drugs demonstrate efficacious properties warranting further investigation with the potential to be implemented as part of the treatment regime for GBM.

Doped Ferrite Nanoparticles Exhibiting Self-Regulating Temperature as Magnetic Fluid Hyperthermia Antitumoral Agents, with Diagnostic Capability in Magnetic Resonance Imaging and Magnetic Particle Imaging.

Simple SummaryHyperthermia, a limited increase in tumor tissue temperature up to a maximum of 45 °C, has long been used in cancer treatment, either as a stand-alone therapy or in conjunction with chemo-/radiotherapy. Among the different physical procedures used, a particularly promising technique is magnetic fluid hyperthermia (MFH): this consists of the local administration of magnetic nanoparticles, followed by the application of alternating magnetic fields. One concern with this technique is the possibility of damage to healthy peritumoral tissue. The present study investigates innovative nanoparticles with self-regulating temperature, which should reduce this risk and thus mark a significant step forward in MFH. In an experimental model of aggressive breast cancer, we demonstrated a substantial reduction of tumor growth rate by using an experimental MFH protocol, transferable to clinical practice. These innovative nanomaterials present the added advantage of allowing non-invasive monitoring of temperature, by magnetic resonance imaging (MRI) and magnetic particle imaging (MPI).This paper reports a comprehensive investigation of a magnetic nanoparticle (MNP), named M55, which belongs to a class of innovative doped ferrite nanomaterials, characterized by a self-limiting temperature. M55 is obtained from M48, an MNP previously described by our group, by implementing an additional purification step in the synthesis. M55, after citrate and glucose coating, is named G-M55. The present study aimed to demonstrate the properties of G-M55 as a diagnostic contrast agent for MRI and magnetic particle imaging (MPI), and as an antitumoral agent in magnetic fluid hyperthermia (MFH). Similar specific absorption rate values were obtained by standard MFH and by an MPI apparatus. This result is of interest in relation to the application of localized MFH by MPI apparatus. We demonstrated the biocompatibility of G-M55 in a triple-negative human breast cancer line (MDA-MB-231), and its efficacy as an MFH agent in the same cell line. We also demonstrated the efficacy of MFH treatment with G-M55 in an experimental model of breast cancer. Overall, our results pave the way for the clinical application of G-M55 as an MFH agent in breast cancer therapy, allowing not only efficient treatment by both standard MFH apparatus and MPI but also temperature monitoring.

Bleomycin-triamcinolone sclerotherapy in the management of propranolol resistant infantile hemangioma of the maxillofacial region: A single arm prospective evaluation of clinical outcome and Doppler ultrasound parameters

IntroductionPropranolol has emerged as a first line agent in the management of hemangiomas. With increased use of propranolol, studies have also focused on relapses following propranolol therapy. Our current study evaluates the role of bleomycin triamcinolone sclerotherapy for the management of propranolol resistant Infantile Hemangioma (IH). We also evaluated the role color Doppler USG for response assessment of sclerotherapy. MethodsPatients with Propranolol resistant (Non- responders/ Partial responders) IHs were included in the study. Patients received intralesional bleomycin at a dose of 0.5 IU/kg followed by intralesional injection of triamcinolone at a dose of 2mg/kg body weight. Clinically patients were grouped into excellent, partial and non responders. Doppler parameters; Resistivity index (RI), Pulsatility index(PI) and Peak systolic velocity (PSV) were used to evaluate the response to sclerotherapy. These parameters were evaluated prior to sclerotherapy and 3 months following completion of therapy. The clinical responses of the patients were compared with the change in Doppler parameters before and after treatment. ResultsA total of 115 participants were considered for analysis; 60.86% had excellent response, 32.17% had partial response and 6.95% had poor response. There was a significant change in terms of RI, PI and PSV in patients who were either excellent or partial responders. Poor responders did not have a significant change in Doppler parameters. Conclusion: Combined bleomycin- triamcinolone sclerotherapy is an effective therapy for the management of propranolol resistant IHs. Doppler parameters RI, PI and PSV are reliable indicators of response in the management of IH.

Anticancer activity, hTERT expression and telomere length analysis in SH-SY5Y neuroblastoma cell lines applied to docetaxel

Neuroblastoma is the most common extracranial solid tumor of infancy in a broad range of clinical courses, ranging from spontaneous regression to fatal progression. Telomere maintenance plays an important role in genome stability and cell proliferation. Telomerase reverse transcriptase (hTERT in humans) is a catalytic subunit of the enzyme telomerase. In this study, it was aimed to determine the anticancer activity of the docetaxel chemotherapeutic agent in neuroblastoma cell line (SH-SY5Y) and to investigate its effect on hTERT gene expression level and telomere length. Molecular docking studies were performed on docetaxel with the crystal structure of telomerase. The electronic properties of docetaxel were calculated using the density functional theory (DFT) method. SH-SY5Y cells were treated for 24, 48 and 72 h with specific concentrations of docetaxel drug ranging from 1 to 100 µg/ml. IC50 doses of docetaxel were determined and administered to SH-SY5Y cells, followed by RNA isolation. hTERT and MYC gene expression levels and telomere length were measured in the docetaxel-treated sample using the RT-PCR method. In addition, theoretical analyzes were made. The IC50 values of docetaxel after 24, 48 and 72 h were 8.32±1.45 μg/ml, 7.67±2.56 μg/ml and 5.51±1.24 μg/ml, respectively. According to the results obtained, docetaxel was found to have the highest activity in 72 h of incubation. It was determined that the docetaxel drug decreased the expression level of the hTERT gene in SH-SY5Y cells. Telomere lengths were significantly reduced in the docetaxel treated SH-SY5Y cell line compared to the control group (p < 0.05). Molecular docking analysis results were in agreement with the experiments. Analysis results indicated a good interaction between docetaxel and the active site of telomerase. The results of this study, reinforced by molecular docking analyzes, might be proved valuable for the development of potent telomerase inhibitors.

Assessment of Oxaliplatin-Loaded Iodine Nanoparticles for Chemoradiotherapy of Human Colorectal Cancer (HT-29) Cells.

Colorectal cancer is highly prevalent worldwide and has significant morbidity and mortality in humans. High-atomic-number nanoparticles such as iodine can act as X-rays absorbers to increase the local dose. The synthesis and fabrication of oxaliplatin-loaded iodine nanoparticles, their characterization, cell toxicity, radiosensitivity, cell apoptosis, and cell cycle assay in human colorectal cancer (HT-29) cells are investigated. Results show that the synthesis of a new iodine nanoparticle, polymerized triiodobenzene coated with chitosan and combined with oxaliplatin as a chemotherapeutic drug, performed well in vitro in an intracellular radiosensitizer as chemoradiotherapy agent in HT-29 cell lines. Findings also show that the INPs alone have no impact on cell cycle development and apoptosis. In contrast, oxaliplatin-loaded INPs along with 2 and 6 MV radiation doses produced more apoptosis. The interaction of INPs with mega-voltage photon energies is the cause of a major radiosensitization enhancement in comparison to radiation alone. Furthermore, results show that INPs may work as radiosensitization nanoprobe agents in the treatment of HT-29 cells due to their effect on increasing radiation dose absorption. Overall, iodine nanoparticles may be used in the treatment of colorectal cancers in clinical studies.

S724 Insurance Companies’ Poor Adherence to ACG/AGA Guidelines for Moderate to Severe Ulcerative Colitis and Crohn’s Disease Management

Introduction: Management of moderate-to-severe ulcerative colitis (UC) and Crohn’s disease (CD) has shifted from step-up therapy to induction of remission with a biologic agent based on disease severity. We sought to assess if current insurance company policies meet the latest guidelines from the ACG and AGA for UC and CD management. Methods: The top 50 insurance companies were searched for publicly available policy information for infliximab, adalimumab, vedolizumab, tofacitinib, and ustekinumab. Data extracted included authors, date of last revision, citation of AGA/ACG guidelines, and policy requirements. Additionally, data regarding need to fail conventional therapy, use of biosimilars, and use of 1st line biologic agents was obtained. The data was compared to the 2018 ACG and 2020 AGA guidelines for UC, and the 2019 ACG and 2021 AGA guidelines for CD. Results: Of the top 50 insurance companies, 48 provided health coverage. Of those, 33 (72.34%) had publicly accessible policies on the coverage of biologics. No authors of the policies were listed. ACG/AGA guidelines were directly quoted or cited in 70.6% of policies. Policies were updated from 1/1/2016 to 5/31/2022. Of the 34 policies that were analyzed, only 58.8% of policies were consistent with the ACG guidelines for CD vs 8.8% to AGA guidelines (Table). Additionally, only 14.7% and 17.7% of policies permitted any first-line biologic therapy in CD and in UC respectively. Conclusion: As of 2022, insurance companies that comprise nearly 80% of the market are yet to adopt the most current guidelines for IBD management. Nearly every insurance company requires the failure of “conventional” therapies such as 5-ASAs, immunomodulators, and corticosteroids. The time required to determine therapeutic failure was inconsistent – ranging from 7 days to 6 months. The large difference between adherence to ACG vs AGA guidance for CD is due to the difference in recommendations for use of an immunomodulator vs anti-TNF as first line agent, as well as length of treatment with corticosteroids before failure (short term therapy vs 3-5 days). The second most common reason for guideline non-adherence was the inability to use immunomodulators concurrently with biologics. Finally, first-line biologic medications were limited primarily to adalimumab or infliximab. Further work is needed to better understand the implications of these inconsistencies between insurance companies and formal medical guidelines on outcomes for patients with moderate-to-severe UC and CD. Table 1. - Adherence to Published Professional Society Guidelines Metric Ulcerative Colitis Crohn's disease ACG Guidelines (2018, 2019) 3/34 (8.8%) 20/34 (58.8%) AGA Guidelines (2020, 2021) 3/34 (8.8%) 2/34 (5.8%) Any first line biologic (infliximab, adalimumab, vedolizumab) 5/34 (14.7%) 6/34 (17.7%) Requirement to fail at least one conventional therapy 31/34 (91%)* 31/34 (91%)** *Certain policies have exceptions including hospitalization for UC.**Certain policies have exceptions including fistulizing disease for CD.

92 (PB082) - RP12146, a novel, selective, and potent small molecule inhibitor of PARP 1/2, synergizes with SOC therapy in preclinical models of solid tumors

Background: Poly (ADP-ribose) polymerase (PARP) activity involves synthesis of Poly-ADP ribose (PAR) polymers that recruit host DNA repair proteins leading to correction of DNA damage and maintenance of cell viability. Several PARP inhibitors have been approved for the treatment of BRCA-mutated ovarian, breast and pancreatic cancer. In addition to BRCA-mutated cancers, therapeutic role of PARP inhibitors as monotherapy agent or in combination with established therapy can be expanded to other types of solid tumors through ‘synthetic lethality’ mechanism. Herein, we describe the efficacy of RP12146 as a single agent and in combination with approved therapies in preclinical models of solid tumors. Material & Methods: Enzymatic potency was evaluated using a PARP Chemiluminescent Activity Assay Kit (BPS biosciences). Cell growth was determined following incubation with RP12146 as a single agent or in combination with approved agents in various solid tumor cell lines. Apoptosis & Cell cycle was evaluated following incubation of cell lines with compound for 48 or 72 h, subsequent staining with Annexin-V-PE and 7-AAD or Propidium Iodide, and analysis by flow cytometry. Anti-tumor potential of RP12146 as a single agent or in combination with chemotherapeutic agents was tested in OVCAR-3 and NCI-H69 Xenografts. Expression of downstream markers were determined in cell lines and xenograft tumor samples by Western blotting. Results: RP12146 inhibited PARP1 (0.6 nM) & PARP2 (0.5 nM) with equal potency with several fold selectivity over the other members of the PARP family. In addition, RP12146 demonstrated selective PARP1-Trapping with ∼20-fold selectivity over PARP2. RP12146 caused a dose-dependent growth inhibition of both BRCA mutant and non-mutant cancer cell lines with GI50 in the range of 0.04 μM to 9.6 μM. Combination of RP12146 with Temozolomide, SN-38, Topotecan, and Lurbinectedin demonstrated either additive or synergistic effects manifested by an inhibition in growth, induction of apoptosis and cell cycle arrest in Breast, Gastric, Glioma, Lung Cancer, and Ovarian cancer cell lines. RP12146 exhibited anti-tumor potential with TGI of 28% and 21.8% as a single agent in OVCAR-3 and NCI-H69 Xenograft model respectively. In NCI-H69 xenograft model, RP12146 in combination with cisplatin exhibited TGI of 69.3%. Conclusion: Data demonstrate the therapeutic potential of RP12146 as single agent and in combination in solid tumors. RP12146 is currently being evaluated in Phase 1 clinical trials in patients with locally advanced or metastatic solid tumors (NCT05002868). No conflict of interest.

93 (PB083) - Preclinical Profile of RP14042, a novel, selective, and potent small molecule inhibitor of PARP7

Background: Poly (ADP-ribose) polymerase (PARP)-7 is a member of PARP family and is classified as monoPARP as it catalyzes the transfer of single units of ADP-ribose (MARylation) onto its substrates to exert its physiological functions. It is involved in several cellular processes, including responses to hypoxia, innate immunity, and regulation of nuclear receptors. In addition, PARP7 is overexpressed/amplified in variety of cancers including Ovarian, Breast, Pancreatic ductal adenocarcinoma (PDAC), Non-small cell lung carcinoma (NSCLC) and Squamous cancers. PARP-7 acts as a negative regulator of nucleic acid sensing in tumor cells leading to suppression of Type I interferon (IFN) response. Preclinical studies have demonstrated that inhibition of PARP-7 restores Type I interferon (IFN) signaling leading to tumor cell growth inhibition and activation of the immune system, thereby contributing to tumor regression. Herein, we describe the preclinical profile of RP14042, a novel, selective, and potent small molecule inhibitor of PARP7. Material & Methods: Enzymatic potency was evaluated using a PARP Chemiluminescent Activity Assay Kit (BPS biosciences). Cell growth was determined following incubation with RP14042 as a single agent in various solid tumor cell lines. Cell cycle was evaluated following incubation of cells with compound for 72 h, subsequent staining with Propidium Iodide, and analysis by flow cytometry. Expression of p-STAT1 and p-IRF3 were determined in NCI-H1373 cell line by Western blotting. Pharmacokinetic properties of the molecule were also evaluated. Results: RP14042 inhibited PARP7 with an IC50 of 10.2 nM with selectivity over other members of the PARP family. Compound caused a dose-dependent growth inhibition of NCI-H1373, a PARP-7 overexpressing, cell line with an GI50 of 0.5 μM without any effect on the growth in low PARP-7 expressing cell lines. Incubation of NCI-H1373 cells with RP14042 caused G0/G1 cell cycle arrest. Inhibition of PARP7 by RP14042 in NCI-H1373 induced STAT1 and IRF3 phosphorylation suggesting restoration of Type I IFN signaling. Pharmacokinetic studies in rodents indicated high oral bioavailability with favorable plasma concentrations relevant for efficacy. Conclusion: Data demonstrate the therapeutic potential of RP14042 in PARP7 overexpressing tumors No conflict of interest.

S3000 Ezetimibe-Induced Autoimmune Hepatitis: An Uncommon Offender

Introduction: Drug-induced autoimmune hepatitis (DIAIH) is a subset of autoimmune hepatitis (AIH) leading to a seropositive hepatitis caused by an inciting drug. Ezetimibe is a cholesterol absorption inhibitor within the intestinal tract that very rarely causes DIAIH. The proposed mechanism of Ezetimibe-induced DIAIH is thought to be due to interactions between drug metabolites and CYP450 within hepatic cells. Statins are another class of medication known to cause hepatotoxicity that are commonly used in combination with ezetimibe for hyperlipidemia. Highlighting cases of drug-related autoimmune hepatitis can be useful as routine liver function testing for statin-induced hepatitis is currently not recommended. Case Description/Methods: This case describes a 67 year old man with hyperlipidemia and Hashimoto’s thyroiditis admitted for abdominal pain and biochemical evidence of hepatitis after starting ezetimibe 6 weeks prior. He had also been on atorvastatin but had been on a stable dose for the previous 7 years. He did not have a history of alcohol, herbal medication or Tylenol use and had entirely normal liver function tests 6 months prior to admission. Admission labs were pertinent for AST of 2159, ALT of 3011, Alkaline phosphatase of 245, total bilirubin of 2.7 (direct bilirubin 1.3), INR of 1.3, and a creatinine kinase of 497. Autoimmune serologies were notable for a positive ANA (1:320) and anti-smooth muscle antibody (1:320). An ultrasound was negative for hepatic lesions or evidence of advanced cirrhosis. Ezetimibe and atorvastatin therapy were discontinued on admission and the patient was eventually discharged after his liver function tests improved. Discussion: Ezetimibe is a common 2nd line agent used for patients with statin-refractory hyperlipidemia which has also been known to cause hepatic injury typically with a latency period of 1-2 months into treatment. When used in combination with a statin the risk of hepatic injury increases. Current guidelines recommend against regular surveillance of liver function tests during statin treatment given that significant hepatotoxicity is exceedingly rare and routine testing did not prevent these events. However, in patients taking both a statin and ezetimibe it may be beneficial to obtain pre-treatment and in-treatment liver function testing towards the end of the latency period, especially if the incidence of ezetimibe-induced autoimmune hepatitis continues to rise.

S3060 Labetalol, a Common Antihypertensive, an Uncommon Cause of Drug-Induced Liver Injury (DILI)

Introduction: Labetalol is an antihypertensive medication (AHM) commonly used as first line agent for non-severe hypertension in pregnancy. We present a case of DILI in a young woman transitioned to labetalol while attempting to conceive. Case Description/Methods: A 35-year-old woman with a h/o hypertension, hashimoto's thyroiditis, anxiety, obesity (BMI 34) was admitted to our hospital for abdominal pain, jaundice, pruritus and aminotransferase (LFT) elevations. She had been admitted previously at an outside facility with nausea, anorexia, and elevated LFTs (ALT 1264, AST 1419, ALP 294, TBili 5) 20 weeks after initiation of labetalol. There were no h/o alcohol abuse or risk factors for hepatitis B/C. Home medications including labetalol were stopped. Imaging studies including MRCP showed no evidence of biliary obstruction. Infectious workup (hepatitis A/B/C/E, CMV, HSV, EBV) and drug/tox screen were negative. A liver biopsy showed acute hepatocellular injury without eosinophils, steatosis, cholestasis or fibrosis; differentials included DILI and infectious hepatitis. LFTs improved, patient was discharged and labetalol restarted. Two weeks later, she presented to our hospital with worsening jaundice. Admission labs: ALT 905, AST 1553, ALP 257, TBili 19.8, INR 1.4 and creatinine 1. On exam, there was no stigmata of chronic liver disease or encephalopathy. Labetalol was discontinued. Infectious, hereditary, metabolic were negative. ANA was positive (1:320) homogenous pattern with mild elevation in IgG. Prednisolone 40 mg daily was started with rapid improvement in LFTs. At 8 week follow up, she was asymptomatic and LFTs were near normal (Tbili 2); prednisolone was tapered. Discussion: Mild to moderate, transient aminotransferase elevations are noted in up to 8% of patients on labetalol. But they are asymptomatic and resolve even with continuation of the medication. Severe liver injury however is rare and rapid resolution typically ensues after cessation of the drug. However acute liver failure, death and OLT have been reported. There are isolated case reports and small case series of labetalol induced DILI, mostly in pregnant women. In a case series, 3 out of 11 patients died and 1 of 5 patients who had liver biopsy had chronic active hepatitis. The mechanism of labetalol hepatic injury is believed to be idiosyncratic. Inadvertent rechallenge has been shown to cause recurrence of DILI. We present this case to increase awareness of labetalol induced DILI as it is the most commonly used AHM in pregnant women.

S3025 Hepatic Sarcoidosis Presenting as Decompensated Liver Cirrhosis

Introduction: Sarcoidosis is a disorder characterized by the formation of non-caseating granulomas in multiple organ systems. While pulmonary manifestation is the most common presentation, hepatic involvement can be seen in many patients. Most patients with liver involvement are asymptomatic; however, some can present with symptoms of liver injury. The presentation of hepatic sarcoidosis can resemble many other disease processes. Since definite diagnostic criteria have not yet been formalized for hepatic sarcoidosis, it can often be challenging for providers to make a prompt diagnosis. Case Description/Methods: We present the case of a 36-year-old female who was evaluated for generalized abdominal pain and left flank pain. On presentation, labs were notable for calcium at 13 mg/dl, ionized calcium at 1.62 mmol/l, PTH at 8 pg/ml, 1,25-dihydoxyvitamin D at 96.4 pg/ml, alkaline phosphatase at 192 IU/L, AST at 51 IU/L and ALT at 47 IU/L. Eight months prior, the patient was admitted to an outside hospital in the setting of hematemesis. EGD with variceal banding was performed, and she was diagnosed with cryptogenic liver cirrhosis. CT A/P demonstrated marked heterogeneity of the liver. MRI of the abdomen showed mild hepatosplenomegaly and “diffuse small areas of altered signal intensity throughout the liver suspicious for diffuse hepatic metastatic disease” (Figure A). The patient just had her first visit with an oncologist prior to her presentation to our hospital. Her physical exam was notable for hepatomegaly, abdominal distension and RUQ tenderness. Her hypercalcemia was treated with fluids. Infectious and autoimmune workup came back negative. Different tumor markers including CA 19-9, CA 27-29 and CA 125 were normal. Liver biopsy showed hepatic parenchyma with numerous non-necrotizing granulomas and associated fibrosis. IL-2R receptor-serum came back elevated at 2916.1 pg/ml. The patient was then started on 20 mg prednisone daily for treatment of hepatic sarcoidosis. Discussion: Hepatic sarcoidosis can have a wide spectrum of clinical presentation, ranging from incidental finding to end-stage liver disease. When a patient is diagnosed with a new hepatic disease, keeping hepatic sarcoidosis on the differential is very important. In case of hepatic sarcoidosis, assessing symptoms of liver involvement and biochemical evidence of cholestasis is crucial. While observation is indicated for asymptomatic liver disease, corticosteroids and/or ursodeoxycholic acid are the first line agents for symptomatic disease.Figure 1.: Hypointense liver lesions on MRI, initially presumed suspicious for diffuse hepatic metastatic disease, later confirmed to be sarcoid lesions.

S2374 Recurrent Strictures in a Patient With Missed Diagnosis of Eosinophilic Esophagitis

Introduction: EoE is an antigen-mediated inflammatory disorder of the esophagus that affects both children and adults. Diagnosis requires symptoms related to esophageal dysfunction and esophageal biopsy showing >15 eosinophils/hpf is confirmatory for diagnosis. Proton pump inhibitors (PPIs) are considered the first line agents for the treatment of EoE, together with elimination diet and topical corticosteroids. Nonadherence to therapy and undiagnosed disease can increase the risk of fibro-stricturing disease and food impactions. Practicing evidence-based guidelines in diagnosing and managing patients with EoE can allow standardization of care and prevent complications in patients with EoE. Case Description/Methods: A 34-year-old man presented with a longstanding history of intermittent dysphagia to solids. He had an emergent esophagogastroduodenoscopy (EGD) for food impaction about 3 years prior to presentation. No biopsies were obtained at that time, and he was prescribed a short course of PPI before discharge. On current presentation, the patient underwent an EGD which showed multiple untraversable strictures requiring dilation (Figure). Biopsy from multiple sites showed >200 eosinophils/hpf, consistent with a diagnosis of eosinophilic esophagitis (EoE). Thereafter, the patient was initiated on PPIs but had to undergo further dilations for recurrent strictures. Topical steroids were subsequently added to the therapy with satisfactory response. Discussion: Once considered rare, EoE is now a well-known entity, with an estimated prevalence of 51.6/100000 in the US. However, variability and deviations from standard therapy continues to persist among gastroenterologists. According to a national survey conducted in 2020 among 240 gastroenterologists, only 24 % were “very familiar” with the EoE guidelines and only 12 % followed EoE guidelines in their clinical practice. The current case underscores the need to standardize clinical practice related to the care of patients with EoE in order to provide quality and evidence-based care and improve overall outcomes.Figure 1.: EGD showed trachealization of the esophagus.

P47 The conundrums of Macrophage activation syndrome (MAS) - when to consider a Haematopoietic Stem Cell Transplant (HSCT)

P47 The conundrums of Macrophage activation syndrome (MAS) - when to consider a Haematopoietic Stem Cell Transplant (HSCT)

Low Use of First-Line Agents for Constipation by Primary Care Providers in Patients Referred to Gastroenterology Clinic for Chronic Constipation

Constipation is a highly prevalent and chronic disorder, reported in anywhere from 24-50% of the adult population. The majority of patients with chronic constipation are treated in a primary care setting rather than by a gastroenterologist. In our clinical experience at University of California San Francisco (UCSF), patients are often referred to a gastroenterology (GI) specialist prior to being trialed on an over-the-counter first line agent for constipation, such as fiber or polyethylene glycol (PEG) 3350, which has been shown to be safe and effective for use in patients with chronic constipation regardless of age.