Liposomal Irinotecan Shows a Larger Therapeutic Index than Non-liposomal Irinotecan in Patient-Derived Xenograft Models of Pancreatic Cancer.

INTRODUCTION: Liposomal irinotecan promotes controlled sustained release of irinotecan (CPT-11), therefore, we hypothesize that the therapeutic index (quantitative measurement of the relative efficacy/safety ratio of a drug) will be higher for liposomal than non-liposomal irinotecan. METHODS: We compared the therapeutic indexes of liposomal and non-liposomal irinotecan in mice bearing subcutaneous patient-derived xenograft (PDX) pancreatic tumors under dosing regimens approximating the clinical setting. Following preliminary drug sensitivity/antitumor activity analyses on three PDX tumor models, one model was selected for analyses of efficacy, biomarker, toxicology, pharmacokinetics in mice receiving liposomal irinotecan (2.5, 10, 50 mg/kg/week) or non-liposomal irinotecan (10, 25, 50 mg/kg/week). The maximum tolerated dose (MTD) for each treatment was 50 mg/kg/week. RESULTS: Using the selected IM-PAN-001 model at the MTD (both treatments, 50 mg/kg/week), antitumor activity, phospho-histone gamma-H2AX protein staining in cancer cell nuclei, histological tumor regression, and plasma levels of CPT-11 and its active metabolite SN-38 after 24 h were greater with liposomal than non-liposomal irinotecan, but tumor SN-38 levels were similar. At the lowest doses assessed, antitumor activity, histological tumor regression, and jejunum and bone marrow toxicity were similar. Based on these findings, liposomal and non-liposomal irinotecan had therapeutic indexes of 20 and 5, respectively. CONCLUSION: This non-clinical study showed a fourfold broader therapeutic index with liposomal than non-liposomal irinotecan in mice bearing IM-PAN-001 PDX pancreatic tumors, even at optimal dosing for the two drugs. These findings support the clinical benefit observed with liposomal irinotecan in patients with pancreatic cancer.

Augmenting Experimental Gastric Cancer Activity of Irinotecan through Liposomal Formulation and Antiangiogenic Combination Therapy.

Gastric adenocarcinoma (GAC) is the third most common cause of cancer-related deaths worldwide. Combination chemotherapy remains the standard treatment for advanced GAC. Liposomal irinotecan (nal-IRI) has improved pharmacokinetics (PK) and drug biodistribution compared with irinotecan (IRI, CPT-11). Angiogenesis plays a crucial role in the progression and metastasis of GAC. We evaluated the antitumor efficacy of nal-IRI in combination with novel antiangiogenic agents in GAC mouse models. Animal survival studies were performed in peritoneal dissemination xenografts. Tumor growth and PK studies were performed in subcutaneous xenografts. Compared with controls, extension in animal survival by nal-IRI and IRI was >156% and >94%, respectively. The addition of nintedanib or DC101 extended nal-IRI response by 13% and 15%, and IRI response by 37% and 31% (MKN-45 xenografts); nal-IRI response by 11% and 3%, and IRI response by 16% and 40% (KATO-III xenografts). Retardation of tumor growth was greater with nal-IRI (92%) than IRI (71%). Nintedanib and DC101 addition tend to augment nal-IRI or IRI response in this model. The addition of antiangiogenic agents enhanced tumor cell proliferation inhibition effects of nal-IRI or IRI. The tumor vasculature was decreased by nintedanib (65%) and DC101 (58%), while nal-IRI and IRI alone showed no effect. PK characterization in GAC xenografts demonstrated that compared with IRI, nal-IRI treatment groups had higher retention, circulation time, and tumor levels of CPT-11 and its active metabolite SN-38. These findings indicate that nal-IRI, alone and in combination with antiangiogenic agents, has the potential for improving clinical GAC therapy.

First-line liposomal irinotecan with oxaliplatin, 5-fluorouracil and leucovorin (NALIRIFOX) in pancreatic ductal adenocarcinoma: A phase I/II study.

BACKGROUND: This open-label, phase I/II study evaluated safety and efficacy for first-line liposomal irinotecan + oxaliplatin + 5-fluorouracil + leucovorin (NALIRIFOX). METHODS: Patients (aged ≥18 years) had locally advanced/metastatic pancreatic ductal adenocarcinoma (mPDAC), with an Eastern Cooperative Oncology Group performance status score of 0/1 and adequate organ function. Primary objectives were to determine the maximum tolerated dose (MTD) and to evaluate safety and tolerability. Treatment-emergent adverse events (TEAEs) were graded using National Cancer Institute Common Terminology Criteria for Adverse Events v4.03. Efficacy end-points included progression-free survival (PFS) and overall survival (OS); disease assessments used Response Evaluation Criteria in Solid Tumors 1.1. RESULTS: The MTD (liposomal irinotecan 50 mg/m2 [free-base equivalent], oxaliplatin 60 mg/m2, 5-fluorouracil 2400 mg/m2, leucovorin 400 mg/m2 every 2 weeks) was based on dose-limiting toxicities and cumulative safety data in four dose-exploration cohorts. The MTD was received by 32 of 56 patients, seven during dose exploration and 25 during dose expansion (median age 58.0 years [range, 39-76], 28 [87.5%] with metastatic disease at diagnosis [29 at study entry], and one receiving study treatment at data cutoff [26 February 2020]). Of these patients, 22 of 32 had grade ≥3 treatment-related TEAEs, most commonly neutropenia (31.3%), febrile neutropenia (12.5%) and hypokalaemia (12.5%); ten had serious treatment-related TEAEs; and three died from TEAEs considered unrelated to treatment. Median PFS and OS were 9.2 (95% CI: 7.69-11.96) and 12.6 (8.74-18.69) months, respectively. CONCLUSION: First-line NALIRIFOX for patients with locally advanced/mPDAC was generally manageable and tolerable. A randomised, controlled phase III study is underway.

Phase I study of liposomal irinotecan in patients with metastatic breast cancer: findings from the expansion phase.

PURPOSE: Metastatic breast cancer (mBC) remains incurable and is associated with low survival rates. This study assessed the efficacy and safety of liposomal irinotecan in heavily pretreated patients with mBC, with or without active brain metastases (BM). METHODS: Following the dose escalation phase and determination of recommended phase 2 dose, the expansion phase of this phase I, open-label, non-randomized study, assigned adult women to cohorts based on mBC subtype: cohort 1, hormone receptor +/human epidermal growth factor receptor 2-; cohort 2, triple-negative breast cancer; or cohort 3, any mBC subtype with active BM. Patients received liposomal irinotecan 50 or 70 mg/m2 free base every 2 weeks. Here, we report secondary outcomes including best overall response (BOR), objective response rate (ORR), and treatment-emergent adverse events (TEAEs). RESULTS: For non-central nervous system (non-CNS) disease across all cohorts (intent-to-treat population, N = 29), the ORR was 34.5% (95% confidence interval: 17.94-54.33), with a BOR of partial response in 10 patients (34.5%), stable disease in five (17.2%), progressive disease in 10 (34.5%); four patients were unevaluable (13.8%). The ORR for the CNS cohort was 30.0% (95% confidence interval: 6.67-65.25) using modified Response Evaluation Criteria in Solid Tumors. Common grade 3 or higher TEAEs were diarrhea (27.6%), nausea (17.2%), fatigue (13.8%), asthenia (10.3%), and hypokalemia (10.3%). Serious treatment-related TEAEs were reported in six patients (20.7%). No treatment-related TEAEs resulted in death. CONCLUSIONS: Liposomal irinotecan monotherapy demonstrated antitumor activity in heavily pretreated patients with mBC, with or without BM. The observed safety profile was consistent with that in previous studies. CLINICAL TRIAL REGISTRATION: Trial registration ID NCT01770353.

MicroRNA-4417 is a tumor suppressor and prognostic biomarker for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer with poor prognosis due to lack of druggable targets such as hormone and growth factor receptors. Therefore, identification of targetable regulators such as miRNAs could provide new avenues for therapeutic applications. Here, we report that the expression of miR-4417 is suppressed during the progression of TNBC cells from non-malignant to the malignant stage. MiR-4417 is localized to chromosome 1p36, a region with high frequency of loss of heterozygosity in multiple cancers, and its biogenesis is DICER-dependent. Low expression of miR-4417 is significantly associated with worse prognosis in TNBC patients, while overexpression of miR-4417 is sufficient to inhibit migration and mammosphere formation of TNBC cells in vitro. Overall, our findings suggest miR-4417 exerts a tumor suppressive effect and thereby could serve as a prognostic biomarker and therapeutic tool against TNBC.

Aberrant transcriptomes and DNA methylomes define pathways that drive pathogenesis and loss of brain laterality/asymmetry in schizophrenia and bipolar disorder.

Although the loss of brain laterality is one of the most consistent modalities in schizophrenia (SCZ) and bipolar disorder (BD), its molecular basis remains elusive. Our limited previous studies indicated that epigenetic modifications are key to the asymmetric transcriptomes of brain hemispheres. We used whole-genome expression microarrays to profile postmortem brain samples from subjects with SCZ, psychotic BD [BD[+]] or non-psychotic BD [BD(-)], or matched controls (10/group) and performed whole-genome DNA methylation (DNAM) profiling of the same samples (3-4/group) to identify pathways associated with SCZ or BD[+] and genes/sites susceptible to epigenetic regulation. qRT-PCR and quantitative DNAM analysis were employed to validate findings in larger sample sets (35/group). Gene Set Enrichment Analysis (GSEA) demonstrated that BMP signaling and astrocyte and cerebral cortex development are significantly (FDR q < 0.25) coordinately upregulated in both SCZ and BD[+], and glutamate signaling and TGFß signaling are significantly coordinately upregulated in SCZ. GSEA also indicated that collagens are downregulated in right versus left brain of controls, but not in SCZ or BD[+] patients. Ingenuity Pathway Analysis predicted that TGFB2 is an upstream regulator of these genes (p = .0012). While lateralized expression of TGFB2 in controls (p = .017) is associated with a corresponding change in DNAM (p ≤ .023), lateralized expression and DNAM of TGFB2 are absent in SCZ or BD. Loss of brain laterality in SCZ and BD corresponds to aberrant epigenetic regulation of TGFB2 and changes in TGFß signaling, indicating potential avenues for disease prevention/treatment.

SDPR functions as a metastasis suppressor in breast cancer by promoting apoptosis.

Metastatic dissemination of breast cancer cells represents a significant clinical obstacle to curative therapy. The loss of function of metastasis suppressor genes is a major rate-limiting step in breast cancer progression that prevents the formation of new colonies at distal sites. However, the discovery of new metastasis suppressor genes in breast cancer using genomic efforts has been slow, potentially due to their primary regulation by epigenetic mechanisms. Here, we report the use of model cell lines with the same genetic lineage for the identification of a novel metastasis suppressor gene, serum deprivation response (SDPR), localized to 2q32-33, a region reported to be associated with significant loss of heterozygosity in breast cancer. In silico metaanalysis of publicly available gene expression datasets suggests that the loss of expression of SDPR correlates with significantly reduced distant-metastasis-free and relapse-free survival of breast cancer patients who underwent therapy. Furthermore, we found that stable SDPR overexpression in highly metastatic breast cancer model cell lines inhibited prosurvival pathways, shifted the balance of Bcl-2 family proteins in favor of apoptosis, and decreased migration and intravasation/extravasation potential, with a corresponding drastic suppression of metastatic nodule formation in the lungs of NOD/SCID mice. Moreover, SDPR expression is silenced by promoter DNA methylation, and as such it exemplifies epigenetic regulation of metastatic breast cancer progression. These observations highlight SDPR as a potential prognostic biomarker and a target for future therapeutic applications.

Characterizing short stature by insulin-like growth factor axis status and genetic associations: results from the prospective, cross-sectional, epidemiogenetic EPIGROW study.

CONTEXT: Serum IGF-I levels are often low in patients with short stature (SS) without defined etiology. Hence, genetic investigations have focused on the GH-IGF-I axis. OBJECTIVE: Our objectives were to characterize IGF-I axis status and search for a broader range of genetic associations in children with SS and normal GH. DESIGN AND SETTING: We conducted a prospective, cross-sectional, epidemiogenetic case-control study in 9 European countries (2008-2010). PARTICIPANTS: Children (n = 275) aged ≥2 years with SS without defined etiology (≤-2.5 height SD score [SDS]) and ≥1 peak GH ≥7 µg/L) were recruited. METHODS: Serum IGF-I, IGF-binding protein-3 (IGFBP-3), and acid-labile subunit (ALS) levels were measured in a central laboratory. Candidate gene exome sequencing was performed in this cohort and ethnicity-matched controls. RESULTS: Serum IGF-I, IGFBP-3, and ALS levels were highly correlated, but there was a discrepancy between prevalence of IGF-I, IGFBP-3, and ALS deficiencies (53%, 30%, and 0.8%, respectively). An insertion-deletion (Indel) on the IGF1 gene (P = 1.2 × 10(-5), Bonferroni-corrected; case vs control frequency: 0.04 vs 0.112), an Indel on NFKB1 (P = 1.36 × 10(-10); case vs control frequency: 0.464 vs 0.272), and 2 single-nucleotide polymorphisms on ZBTB38 (P < 2.3 × 10(-6)) were associated with SS. At P < 10(-4), single-nucleotide polymorphisms on genes related to protein kinase regulation, MAPK, and Fanconi pathways were also associated with SS. CONCLUSIONS: IGF-I deficiency is a common feature in SS without defined etiology; an Indel in the IGF1 gene was associated with SS. However, genes involved in transcriptional regulation (NFKB1 and ZBTB38) and growth factor signaling were also associated, providing further candidates for genetic investigations on individual patients.

Smad signaling is required to maintain epigenetic silencing during breast cancer progression.

Breast cancer progression is associated with aberrant DNA methylation and expression of genes that control the epithelial-mesenchymal transition (EMT), a critical step in malignant conversion. Although the genes affected have been studied, there is little understanding of how aberrant activation of the DNA methylation machinery itself occurs. Using a breast cancer cell-based model system, we found that cells that underwent EMT exhibited overactive transforming growth factor beta (TGFbeta) signaling and loss of expression of the CDH1, CGN, CLDN4, and KLK10 genes as a result of hypermethylation of their corresponding promoter regions. Based on these observations, we hypothesized that activated TGFbeta-Smad signaling provides an "epigenetic memory" to maintain silencing of critical genes. In support of this hypothesis, disrupting Smad signaling in mesenchymal breast cancer cells resulted in DNA demethylation and reexpression of the genes identified. This epigenetic reversal was accompanied by an acquisition of epithelial morphology and a suppression of invasive properties. Notably, disrupting TGFbeta signaling decreased the DNA binding activity of DNA methyltransferase DNMT1, suggesting that failure to maintain methylation of newly synthesized DNA was the likely cause of DNA demethylation. Together, our findings reveal a hyperactive TGFbeta-TGFbetaR-Smad2 signaling axis needed to maintain epigenetic silencing of critical EMT genes and breast cancer progression.

hBub1 negatively regulates p53 mediated early cell death upon mitotic checkpoint activation.

Our previous studies showed that the depletion of the outer kinetochore protein hBub1 upon activation of spindle assembly checkpoint (SAC) primarily triggers early cell death mediated by p53 rather than aneuploidy. Here, we report that phosphorylation of p53 at Ser37 is critical for proapoptotic activity upon SAC activation. Furthermore, we show that p53 physically interacts with hBub1 at kinetochores in response to mitotic spindle damage suggesting a direct role for hBub1 in the suppression of p53 mediated cell death. This observation is further substantiated by the inhibition of p53 mediated transactivation of the proapoptotic target genes, PUMA and BAX, by hBub1 in SAC activated cells. In summary, our data from these and our previous studies strongly suggest that in response to SAC activation, hBub1 acts as a negative regulator of p53 mediated early cell death in a novel checkpoint pathway. On the translational medicine front, it is tempting to speculate that by disabling hBub1 in p53 proficient cancer cells, apoptosis may be induced as a therapeutic approach to eradicate the tumor cells.

hBub1 deficiency triggers a novel p53 mediated early apoptotic checkpoint pathway in mitotic spindle damaged cells.

It has been universally believed that spindle assembly checkpoint (SAC) proteins which include the kinetochore proteins are involved in monitoring the faithful segregation of sister chromatids during cell division and hence defects in these proteins result in aneuploidy. Furthermore, there are multiple sources of experimental data to suggest that a defect in p53 can also promote genomic instability leading to aneuploidy. Despite these observations, a molecular basis for the prevention of aneuploidy to maintain genomic integrity upon activation of SAC has largely remained elusive. In this report, we demonstrate a novel mechanism for the maintenance of a balance between cell survival and apoptosis upon activation of SAC. We found that depletion of the outer kinetochore protein hBub1 upon activation of SAC primarily triggers early cell death mediated by p53. This phenomenon is further supported by the upregulation of p53 downstream pro-apoptotic genes, BAX and PUMA as well as a corresponding increase in the cleavage products of PARP and caspase 3, markers of apoptosis, upon depletion of hBub1 in SAC activated cells. On the other hand, as expected, concomitant loss of both hBub1 and p53 resulted in disabling of the p53 mediated cell death pathway leading to the accumulation of cells with aneuploidy/polyploidy.

Differential DNA hypermethylation of critical genes mediates the stage-specific tobacco smoke-induced neoplastic progression of lung cancer.

Promoter DNA methylation status of six genes in samples derived from 27 bronchial epithelial cells and matching blood samples from 22 former/current smokers and five nonsmokers as well as 49 primary non-small cell lung cancer samples with corresponding blood controls was determined using methylation-specific PCR (MSP). Lung tumor tissues showed a significantly higher frequency of promoter DNA methylation in p16, MGMT, and DAPK (P < 0.05; Fisher's exact test). p16 promoter DNA methylation in tumors was observed at consistently higher levels when compared with all the other samples analyzed (P = 0.001; Fisher's exact test). ECAD and DAPK exhibited statistically insignificant differences in their levels of DNA methylation among the tumors and bronchial epithelial cells from the smokers. Interestingly, similar levels of methylation were observed in bronchial epithelial cells and corresponding blood from smokers for all four genes (ECAD, p16, MGMT, and DAPK) that showed smoking/lung cancer-associated methylation changes. In summary, our data suggest that targeted DNA methylation silencing of ECAD and DAPK occurs in the early stages and that of p16 and MGMT in the later stages of lung cancer progression. We also provide preliminary evidence that peripheral lymphocytes could potentially be used as a surrogate for bronchial epithelial cells to detect altered DNA methylation in smokers.

Loss of heterozygosity patterns provide fingerprints for genetic heterogeneity in multistep cancer progression of tobacco smoke-induced non-small cell lung cancer.

Dilution end point loss of heterozygosity (LOH) analysis, a novel approach for the analysis of LOH, was used to evaluate allelic losses with the use of 21 highly polymorphic microsatellite markers at nine chromosomal sites most frequently affected in smoking-related non-small cell lung cancers. Allelotyping was done for bronchial epithelial cells and matching blood samples from 23 former and current smokers and six nonsmokers as well as in 33 adenocarcinomas and 25 squamous cell carcinomas (SCC) and corresponding matching blood from smokers. Major conclusions from these studies are as follows: (a) LOH at chromosomal sites 8p, 9p, 11q, and 13q (P >0.05, Fisher's exact test) are targeted at the early stages, whereas LOH at 1p, 5q, 17p, and 18q (P <0.05, Fisher's exact test) occur at the later stages of non-small cell lung cancer progression; (b) LOH at 1p, 3p, 5q, 8p, 9p, 11q, 13q, 17p, and 18q occurs in over 45% of the tobacco smokers with SCC and adenocarcinoma; (c) compared with bronchial epithelial cells from smokers, there is a significantly higher degree of LOH at 1p, 5q, and 18q in adenocarcinoma and at 1p, 3p, and 17p in SCC (P <0.05, Fisher's exact test). We propose that lung cancer progression induced by tobacco smoke occurs in a series of target gene inactivations/activations in defined modules of a global network. The gatekeeper module consists of multiple alternate target genes, which is inclusive of but not limited to genes localized to chromosomal loci 8p, 9p, 11q, and 13q.

Histone deacetylases: unique players in shaping the epigenetic histone code.

The epigenome is defined by DNA methylation patterns and the associated posttranslational modifications of histones. This histone code determines the expression status of individual genes dependent upon their localization on the chromatin. The silencing of gene expression is associated with deacetylated histones, which are often found to be associated with regions of DNA methylation as well as methylation at the lysine 4 residue of histone 3. In contrast, the activation of gene expression is associated with acetylated histones and methylation at the lysine 9 residue of histone 3. The histone deactylases play a major role in keeping the balance between the acetylated and deacetylated states of chromatin. Histone deacetylases (HDACs) are divided into three classes: class I HDACs (HDACs 1, 2, 3, and 8) are similar to the yeast RPD3 protein and localize to the nucleus; class II HDACs (HDACs 4, 5, 6, 7, 9, and 10) are homologous to the yeast HDA1 protein and are found in both the nucleus and cytoplasm; and class III HDACs form a structurally distinct class of NAD-dependent enzymes that are similar to the yeast SIR2 proteins. Since inappropriate silencing of critical genes can result in one or both hits of tumor suppressor gene (TSG) inactivation in cancer, theoretically the reactivation of affected TSGs could have an enormous therapeutic value in preventing and treating cancer. Indeed, several HDAC inhibitors are currently being developed and tested for their potency in cancer chemotherapy. Importantly, these agents are also potentially applicable to chemoprevention if their toxicity can be minimized. Despite the toxic side effects and lack of specificity of some of the inhibitors, progress is being made. With the elucidation of the structures, functions and modes of action of HDACs, finding agents that may be targeted to specific HDACs and potentially reactivate expression of only a defined set of affected genes in cancer will be more attainable.

Loss of heterozygosity as a predictor to map tumor suppressor genes in cancer: molecular basis of its occurrence.

High frequency of chromosomal deletions elicited as losses of heterozygosity is a hallmark of genomic instability in cancer. Functional losses of tumor suppressor genes caused by loss of heterozygosity at defined regions during clonal selection for growth advantage define the minimally lost regions as their likely locations on chromosomes. Loss of heterozygosity is elicited at the molecular or cytogenetic level as a deletion, a gene conversion, single or double homologous and nonhomologous mitotic recombinations, a translocation, chromosome breakage and loss, chromosomal fusion or telomeric end-to-end fusions, or whole chromosome loss with or without accompanying duplication of the retained chromosome. Because of the high level of specificity, loss of heterozygosity has recently become invaluable as a marker for diagnosis and prognosis of cancer. The molecular defects for the occurrence of loss of heterozygosity are derived from disabled caretaker genes, which protect the integrity of DNA, or chromosome segregator genes, which mediate faithful chromosome disjunction.