Pyrvinium Pamoate Use in a B cell Acute Lymphoblastic Leukemia Model of the Bone Tumor Microenvironment.

PURPOSE: Pyrvinium pamoate (PP) is an anthelmintic drug that has been found to have anti-cancer activity in several cancer types. In the present study, we evaluated PP for potential anti-leukemic activity in B cell acute lymphoblastic leukemia (ALL) cell lines, in an effort to evaluate the repurposing potential of this drug in leukemia. METHODS: ALL cells were treated with PP at various concentrations to determine its effect on cell proliferation. Metabolic function was tested by evaluating Extracellular Acidification Rate (ECAR) and Oxygen Consumption Rate (OCR). Lastly, 3D spheroids were grown, and PP was reformulated into nanoparticles to evaluate distribution effectiveness. RESULTS: PP was found to inhibit ALL proliferation, with varied selectivity to different ALL cell subtypes. We also found that PP's cell death activity was specific for leukemic cells, as primary normal immune cells were resistant to PP-mediated cell death. Metabolic studies indicated that PP, in part, inhibits mitochondrial oxidative phosphorylation. To increase the targeting of PP to a hypoxic bone tumor microenvironment (BTME) niche, we successfully encapsulated PP in a nanoparticle drug delivery system and demonstrated that it retained its anti-leukemic activity in a hemosphere assay. CONCLUSION: We have demonstrated that PP is a novel therapeutic lead compound that counteracts the respiratory reprogramming found in refractory ALL cells and can be effectively formulated into a nanoparticle delivery system to target the BTME.

The MitoNEET Ligand NL-1 Mediates Antileukemic Activity in Drug-Resistant B-Cell Acute Lymphoblastic Leukemia.

Disease relapse in B-cell acute lymphoblastic leukemia (ALL), either due to development of acquired resistance after therapy or because of de novo resistance, remains a therapeutic challenge. In the present study, we have developed a cytarabine (Ara-C)-resistant REH cell line (REH/Ara-C) as a chemoresistance model. REH/Ara-C 1) was not crossresistant to vincristine or methotrexate; 2) showed a similar proliferation rate and cell surface marker expression as parental REH; 3) demonstrated decreased chemotaxis toward bone marrow stromal cells; and 4) expressed higher transcript levels of cytidine deaminase (CDA) and mitoNEET (CISD1) than the parental REH cell line. Based on these findings, we tested NL-1, a mitoNEET inhibitor, which induced a concentration-dependent decrease in cell viability with a comparable IC50 value in REH and REH/Ara-C. Furthermore, NL-1 decreased cell viability in six different ALL cell lines and showed inhibitory activity in a hemosphere assay. NL-1 also impaired the migratory ability of leukemic cells, irrespective of the chemoattractant used, in a chemotaxis assay. More importantly, NL-1 showed specific activity in inducing death in a drug-resistant population of leukemic cells within a coculture model that mimicked the acquired resistance and de novo resistance observed in the bone marrow of relapsed patients. Subsequent studies indicated that NL-1 mediates autophagy, and inhibition of autophagy partially decreased NL-1-induced tumor cell death. Finally, NL-1 showed antileukemic activity in an in vivo mouse ALL model. Taken together, our study demonstrates that mitoNEET has potential as a novel antileukemic drug target in treatment refractory or relapsed ALL.

Novel compounds that target lipoprotein lipase and mediate growth arrest in acute lymphoblastic leukemia.

Over the past decade, the therapeutic strategies employed to treat B-precursor acute lymphoblastic leukemia (ALL) have been progressively successful in treating the disease. Unfortunately, the treatment associated dyslipidemia, either acute or chronic, is very prevalent and a cause for decreased quality of life in the surviving patients. To overcome this hurdle, we tested a series of cylopropanecarboxamides, a family demonstrated to target lipid metabolism, for their anti-leukemic activity in ALL. Several of the compounds tested showed anti-proliferative activity, with one, compound 22, inhibiting both Philadelphia chromosome negative REH and Philadelphia chromosome positive SupB15 ALL cell division. The novel advantage of these compounds is the potential synergy with standard chemotherapeutic agents, while concomitantly blunting the emergence of dyslipidemia. Thus, the cylopropanecarboxamides represent a novel class of compounds that can be potentially used in combination with the present standard-of-care to limit treatment associated dyslipidemia in ALL patients.

Chemotherapeutic Activity of Pitavastatin in Vincristine Resistant B-Cell Acute Lymphoblastic Leukemia.

B-cell acute lymphoblastic leukemia (ALL) is derived from an accumulation of malignant, immature B cells in the bone marrow and blood. Relapse due, in part, to the emergence of tumor cells that are resistant to front line standard chemotherapy is associated with poor patient outcomes. This challenge highlights the need for new treatment strategies to eliminate residual chemoresistant tumor cells. Based on the use of pitavastatin in acute myeloid leukemia (AML), we evaluated its efficacy in an REH ALL cell line derived to be resistant to vincristine. We found that pitavastatin inhibited the proliferation of both parental and vincristine-resistant REH tumor cells at an IC50 of 449 nM and 217 nM, respectively. Mitochondrial bioenergetic assays demonstrated that neither vincristine resistance nor pitavastatin treatment affected cellular oxidative phosphorylation, beta-oxidation, or glycolytic metabolism in ALL cells. In a co-culture model of ALL cells with bone marrow stromal cells, pitavastatin significantly decreased cell viability more robustly in the vincristine-resistant ALL cells compared with their parental controls. Subsequently, NSG mice were used to develop an in vivo model of B-cell ALL using both parental and vincristine-resistant ALL cells. Pitavastatin (10 mg/kg i.p.) significantly reduced the number of human CD45+ REH ALL cells in the bone marrow of mice after 4 weeks of treatment. Mechanistic studies showed that pitavastatin treatment in the vincristine-resistant cells led to apoptosis, with increased levels of cleaved PARP and protein-signaling changes for AMP-activated protein kinase/FoxO3a/Puma. Our data suggest the possible repurposing of pitavastatin as a chemotherapeutic agent in a model of vincristine-resistant B-cell ALL.

Characterization of complex fluvial-deltaic deposits in Northeast India using Poisson impedance inversion and non-parametric statistical technique.

Characterizing complex fluvial-deltaic deposits is a challenging task for finding hydrocarbon discoveries. We described a methodology for predicting the hydrocarbon zones from complex well-log and prestack seismic data. In this current study, data analysis involves an integrated framework based on Simultaneous prestack seismic inversion (SPSI), target correlation coefficient analysis (TCCA), Poisson impedance inversion, and non-parametric statistical analysis, and Bayesian classification. First, seismic elastic attributes from prestack seismic data were estimated. They can provide the spatial distribution of petrophysical properties of seismic data. Then target correlation coefficient analysis (TCCA) was estimated roration factor "c" from well-log data. Using the seismic elastic attributes and rotation factor "c", Poisson impedance inversion was performed to predict the Poisson impedance volume. Finally, Bayesian classification integrated the Poisson impedance volume with non-parametric probabilistic density functions (PDFs) to estimate the spatial distribution of lithofacies. Despite complex characteristics in the elastic properties, the current study successfully delineated the complex fluvial-details deposits. These results were verified with conventional findings through numerical analysis.

Pitavastatin Is Anti-Leukemic in a Bone Marrow Microenvironment Model of B-Lineage Acute Lymphoblastic Leukemia.

The lack of complete therapeutic success in the treatment of B-cell acute lymphoblastic leukemia (ALL) has been attributed, in part, to a subset of cells within the bone marrow microenvironment that are drug resistant. Recently, the cholesterol synthesis inhibitor, pitavastatin (PIT), was shown to be active in acute myeloid leukemia, prompting us to evaluate it in our in vitro co-culture model, which supports a chemo-resistant ALL population. We used phospho-protein profiling to evaluate the use of lipid metabolic active compounds in these chemo-resistant cells, due to the up-regulation of multiple active survival signals. In a co-culture with stromal cells, a shift towards anabolic processes occurred, which was further confirmed by assays showing increased lipid content. The treatment of REH leukemia cells with pitavastatin in the co-culture model resulted in significantly higher leukemic cell death than exposure to the standard-of-care chemotherapeutic agent, cytarabine (Ara-C). Our data demonstrates the use of pitavastatin as a possible alternative treatment strategy to improve patient outcomes in chemo-resistant, relapsed ALL.

Recurrent DHA nephropathy in renal allograft-revisiting clinicopathological aspects of a rare entity.

BACKGROUND: Adenine phosphoribosyltransferase (APRT) enzyme deficiency is a rare autosomal recessive disorder of purine metabolism affecting mainly the kidneys. It can present at any age with varying degrees of acute and chronic renal damage. Though xanthine dehydrogenase inhibitors offer effective control over the disease process, delay in diagnosis and treatment often lead to compromised function of native and even graft kidneys. METHODS: We have done a retrospective search of records of renal biopsies reported at our center during the 5-year period from 2014 to 2018 to identify biopsies with 2,8-dihydroxyadenine crystal deposits. The demographic, clinical, and histopathological findings in these cases were studied and reviewed in the light of available literature. RESULTS: Of 9059 renal biopsies received during the study period, 3 cases had the rare 2,8- dihydroxyadenine (DHA) crystals. All of them were diagnosed for the first time on allograft biopsies. CONCLUSION: A high index of clinical suspicion together with the characteristic microscopic appearance of crystals on renal biopsy and urine microscopy can clinch the diagnosis of this rare disease. Hence, improving awareness about this entity among clinicians and pathologists is extremely important.

Co-culture model of B-cell acute lymphoblastic leukemia recapitulates a transcription signature of chemotherapy-refractory minimal residual disease.

B-cell acute lymphoblastic leukemia (ALL) is characterized by accumulation of immature hematopoietic cells in the bone marrow, a well-established sanctuary site for leukemic cell survival during treatment. While standard of care treatment results in remission in most patients, a small population of patients will relapse, due to the presence of minimal residual disease (MRD) consisting of dormant, chemotherapy-resistant tumor cells. To interrogate this clinically relevant population of treatment refractory cells, we developed an in vitro cell model in which human ALL cells are grown in co-culture with human derived bone marrow stromal cells or osteoblasts. Within this co-culture, tumor cells are found in suspension, lightly attached to the top of the adherent cells, or buried under the adherent cells in a population that is phase dim (PD) by light microscopy. PD cells are dormant and chemotherapy-resistant, consistent with the population of cells that underlies MRD. In the current study, we characterized the transcriptional signature of PD cells by RNA-Seq, and these data were compared to a published expression data set derived from human MRD B-cell ALL patients. Our comparative analyses revealed that the PD cell population is markedly similar to the MRD expression patterns from the primary cells isolated from patients. We further identified genes and key signaling pathways that are common between the PD tumor cells from co-culture and patient derived MRD cells as potential therapeutic targets for future studies.

Signaling networks associated with BCR-ABL-dependent transformation.

BACKGROUND: The fusion protein BCR-ABL results in constitutive tyrosine kinase activity. It also affects downstream targets as well as the subcellular location of the normally tightly regulated Abl tyrosine kinase. METHODS: The authors review the current knowledge concerning the signaling networks associated with BCR-ABL-dependent transformation. RESULTS: Although BCR-ABL is considered a single genetic change, the dysregulated tyrosine kinase activates a network of signals that contributes to cytokine-independent growth, resistance to apoptosis, and genetic instability. CONCLUSIONS: The effectiveness of BCR-ABL-dependent transformation of hematopoietic stem cells is due not to a single pathway but rather to the culmination of a network of signaling pathways.

Stat3 contributes to resistance toward BCR-ABL inhibitors in a bone marrow microenvironment model of drug resistance.

Imatinib mesylate is a potent, molecularly targeted therapy against the oncogenic tyrosine kinase BCR-ABL. Although imatinib mesylate has considerable efficacy against chronic myeloid leukemia (CML), advanced-stage CML patients frequently become refractory to this agent. The bone marrow is the predominant microenvironment of CML and is a rich source of both soluble factors and extracellular matrices, which may influence drug response. To address the influence of the bone marrow microenvironment on imatinib mesylate sensitivity, we used an in vitro bone marrow stroma model. Our data show culturing K562 cells, in bone marrow stroma-derived conditioned medium (CM), is sufficient to cause resistance to BCR-ABL inhibitors. Drug resistance correlated with increased pTyrStat3, whereas no increases in pTyrStat5 was noted. Moreover, resistance was associated with increased levels of the Stat3 target genes Bcl-xl, Mcl-1, and survivin. Finally, reducing Stat3 levels with small interfering RNA sensitized K562 cells cultured in CM to imatinib mesylate-induced cell death. Importantly, Stat3 dependency was specific for cells grown in CM, as reducing Stat3 levels in regular growth conditions had no effect on imatinib mesylate sensitivity. Together, these data support a novel mechanism of BCR-ABL-independent imatinib mesylate resistance and provides preclinical rationale for using Stat3-inhibitors to increase the efficacy of imatinib mesylate within the context of the bone marrow microenvironment.

Crystal structure of the mitochondrial protein mitoNEET bound to a benze-sulfonide ligand.

MitoNEET (gene cisd1) is a mitochondrial outer membrane [2Fe-2S] protein and is a potential drug target in several metabolic diseases. Previous studies have demonstrated that mitoNEET functions as a redox-active and pH-sensing protein that regulates mitochondrial metabolism, although the structural basis of the potential drug binding site(s) remains elusive. Here we report the crystal structure of the soluble domain of human mitoNEET with a sulfonamide ligand, furosemide. Exploration of the high-resolution crystal structure is used to design mitoNEET binding molecules in a pilot study of molecular probes for use in future development of mitochondrial targeted therapies for a wide variety of metabolic diseases, including obesity, diabetes and neurodegenerative diseases such as Alzheimer's and Parkinson's disease.

A novel high-throughput screening system identifies a small molecule repressive for matrix metalloproteinase-9 expression.

Aberrant gene expression is one of the driving forces for cancer progression and is considered an ideal target for chemical intervention. Although emerging bioluminescence reporter systems allow high-throughput searches for small molecules regulatory for gene expression, frequent silencing of reporter genes by epigenetic mechanisms hinders wide application of this drug discovery strategy. Here we report a novel system that directs the integration of a promoter-reporter construct to an open chromosomal location by Flp-mediated homologous recombination, thereby overcoming reporter-gene silencing. Using this system, we have screened more than 8000 compounds in the DIVERSet chemical library for repressors of a matrix metalloproteinase-9 (MMP-9) promoter and identified 5-methyl-2-(4-methylphenyl)-1H-benzimidazole (MPBD) inhibitory for MMP-9 gene expression. Consistent with this effect, MPBD inhibits MMP-9-dependent invasion of UMSCC-1 oral cancer cells, preosteoclast migration, and receptor activator of nuclear factor-kappaB ligand-induced osteoclast activity over concentration ranges that repressed MMP-9 expression. Mechanistic studies indicated that MPBD antagonizes AP-1 function by inhibiting its transactivation activity. We conclude that the Flp-mediated homologous recombination system to direct reporter integration into open chromatin regions represents a novel strategy allowing for the development of high-throughput systems screening for lead compounds targeting aberrant gene expression in cancer.

Single-fraction radiation: A promising adjuvant therapy to prevent keloid recurrence.

INTRODUCTION: Keloids are characterized by collection of atypical fibroblasts with excessive deposition of extracellular matrix components. Keloids are prone to high recurrence (50%-80%) with unimodality treatment. Radiation is a promising approach among the adjuvant modalities in vogue though consensus is lacking on dose-fractionation schedule. AIM: The present study aimed to analyze the efficacy of single-fraction high-dose adjuvant radiotherapy to prevent keloid recurrence. MATERIALS AND METHODS: Details of patients treated for keloids using external beam radiation therapy from January 2011 to December 2016 were retrieved from electronic medical records and radiation therapy charts and analyzed. RESULTS: Thirty-seven keloid lesions in thirty patients were analyzed. Keloids received radiation within 24-72 h postsurgery using 6 MeV electron beam. 45.9% of keloids were in the chest wall. Dose ranged between 5 Gy and 12 Gy in 1-3 fractions. Eight Gy was used in 78.4%. The single fraction was preferred in 91.9%. Good cosmesis was achieved in all except three who had wound dehiscence. Median follow-up was 32.67 months. 16.2% had recurrence. Median time to recur was 13.6 months, and median recurrence-free interval 21.23 months. Among those who received 8 Gy single fraction, 73.4% remained recurrence-free at 5 years. CONCLUSION: Albeit a retrospective analysis, ours is the only study in literature offering 8 Gy single dose, using electrons, as a postoperative adjuvant treatment to prevent recurrence in keloids. Our recurrence rates were similar to that quoted in published series. This hence can be validated in further studies as it is cosmetically acceptable, safe, painless, and cost-effective with good patient compliance.

Combination of cabazitaxel and plicamycin induces cell death in drug resistant B-cell acute lymphoblastic leukemia.

Bone marrow microenvironment mediated downregulation of BCL6 is critical for maintaining cell quiescence and modulating therapeutic response in B-cell acute lymphoblastic leukemia (ALL). In the present study, we have performed a high throughput cell death assay using BCL6 knockdown REH ALL cell line to screen a library of FDA-approved oncology drugs. In the process, we have identified a microtubule inhibitor, cabazitaxel (CAB), and a RNA synthesis inhibitor, plicamycin (PLI) as potential anti-leukemic agents. CAB and PLI inhibited cell proliferation in not only the BCL6 knockdown REH cell line, but also six other ALL cell lines. Furthermore, combination of CAB and PLI had a synergistic effect in inhibiting proliferation in a cytarabine-resistant (REH/Ara-C) ALL cell line. Use of nanoparticles for delivery of CAB and PLI demonstrated that the combination was very effective when tested in a co-culture model that mimics the in vivo bone marrow microenvironment that typically supports ALL cell survival and migration into protective niches. Furthermore, exposure to PLI inhibited SOX2 transcription and exposure to CAB inhibited not only Mcl-1 expression but also chemotaxis in ALL cells. Taken together, our study demonstrates the utility of concomitantly targeting different critical regulatory pathways to induce cell death in drug resistant ALL cells.

Actinomycin D Down-regulates SOX2 Expression and Induces Death in Breast Cancer Stem Cells.

BACKGROUND/AIM: One of the major hurdles in the treatment of breast cancers is the inability of anti-cancer drugs to eliminate the breast cancer stem cells (BCSCs) population, which leads to disease relapse. The dearth in anti-cancer drugs that target BCSCs can be attributed to the absence of in vitro screening models that can not only recapitulate the tumor microenvironment consisting of BCSCs but also preserve the 3-dimensional (3D) architecture of in vivo tumors. MATERIALS AND METHODS: In our present study, we have developed a 3D cell culture system that shows: (i) enrichment of BCSCs, (ii) increased drug resistance, and (iii) generation of hypoxic conditions similar to tumors. RESULTS: Using this model, we were able to screen a FDA-approved diversity set and identify as well as validate actinomycin D as a potential anti-breast cancer agent. Interestingly, we show that actinomycin D specifically targets and down-regulates the expression of the stem cell transcription factor, Sox-2. Additionally, down-regulation of Sox-2 leads to depletion of the stem-cell population resulting in the inability of breast cancer cells to initiate tumor progression. CONCLUSION: This study demonstrates the utility of an in vivo-like 3D cell culture system for the identification and validation of anti-cancer agents that will have a better probability of success in the clinic.

Stable and Potent Selenomab-Drug Conjugates.

Selenomabs are engineered monoclonal antibodies with one or more translationally incorporated selenocysteine residues. The unique reactivity of the selenol group of selenocysteine permits site-specific conjugation of drugs. Compared with other natural and unnatural amino acid and carbohydrate residues that have been used for the generation of site-specific antibody-drug conjugates, selenocysteine is particularly reactive, permitting fast, single-step, and efficient reactions under near physiological conditions. Using a tailored conjugation chemistry, we generated highly stable selenomab-drug conjugates and demonstrated their potency and selectivity in vitro and in vivo. These site-specific antibody-drug conjugates built on a selenocysteine interface revealed broad therapeutic utility in liquid and solid malignancy models.

A bisanthracycline (WP631) represses uPAR gene expression and cell migration of RKO colon cancer cells by interfering with transcription factor binding to a chromatin-accessible -148/-124 promoter region.

The urokinase receptor (uPAR), transcriptionally activated in several cancers, contributes to tumor progression by promoting cell migration and proteolysis, and repressing expression of this gene could be of therapeutic utility. Indeed, targeting regulatory element(s) in the promoter may represent an efficient means for reducing expression because only two alleles have to be neutralized. We previously identified the -148/-124 promoter region, bound with Sp1 and Sp3, as regulatory for uPAR expression in vitro. The purpose of this study was twofold: to determine (a) the accessibility of this region in its natural chromatin setting and (b) the efficacy of WP631, a bisintercalator favoring GC-rich DNA sequences, in repressing endogenous uPAR expression in RKO colon cancer cells. In these cells, DNaseI hypersensitivity, genomic footprinting, and chromatin immunoprecipitation experiments revealed that the -148/-124 uPAR promoter region was accessible in chromatin and bound with Sp1, thus validating it as a therapeutic target. WP631 treatment competed for transcription factor binding to this regulatory region and reduced uPAR mRNA/protein. However, a chemically related compound (WP629), with low DNA binding affinity, failed to diminish uPAR protein amount. GAPDH mRNA level was only modestly affected by WP631, arguing against the possibility that this bisanthracycline universally represses expression of GC-rich promoter-driven genes. Further, uPAR function, as assessed by migration of cells across a vitronectin-coated filter, was attenuated with WP631. Thus, we have shown that the chromatinized -148/-124 regulatory region of the uPAR promoter is accessible to small molecules and that WP631, which disrupts the interaction of DNA binding proteins with this region, diminishes uPAR expression and function.

MTI-101 (cyclized HYD1) binds a CD44 containing complex and induces necrotic cell death in multiple myeloma.

Our laboratory recently reported that treatment with the d-amino acid containing peptide HYD1 induces necrotic cell death in multiple myeloma cell lines. Because of the intriguing biological activity and promising in vivo activity of HYD1, we pursued strategies for increasing the therapeutic efficacy of the linear peptide. These efforts led to a cyclized peptidomimetic, MTI-101, with increased in vitro activity and robust in vivo activity as a single agent using two myeloma models that consider the bone marrow microenvironment. MTI-101 treatment similar to HYD1 induced reactive oxygen species, depleted ATP levels, and failed to activate caspase-3. Moreover, MTI-101 is cross-resistant in H929 cells selected for acquired resistance to HYD1. Here, we pursued an unbiased chemical biology approach using biotinylated peptide affinity purification and liquid chromatography/tandem mass spectrometry analysis to identify binding partners of MTI-101. Using this approach, CD44 was identified as a predominant binding partner. Reducing the expression of CD44 was sufficient to induce cell death in multiple myeloma cell lines, indicating that multiple myeloma cells require CD44 expression for survival. Ectopic expression of CD44s correlated with increased binding of the FAM-conjugated peptide. However, ectopic expression of CD44s was not sufficient to increase the sensitivity to MTI-101-induced cell death. Mechanistically, we show that MTI-101-induced cell death occurs via a Rip1-, Rip3-, or Drp1-dependent and -independent pathway. Finally, we show that MTI-101 has robust activity as a single agent in the SCID-Hu bone implant and 5TGM1 in vivo model of multiple myeloma.

Nail-patella syndrome-a novel mutation in the LMX1B gene.

Nail-patella syndrome (NPS) is an autosomal-dominant pleiotropic disorder characterized by dyplasia of finger nails, skeletal anomalies and frequently renal disease. In the reported case, genetic analysis revealed a new missense mutation in the homeodomain of LMX1B, presumed to abolish DNA binding (c.725T>C, p.Val242Ala). A missense mutation at codon 725 was identified, where thymine was replaced by cytosine which led to the replacement of valine by alanine at position 242. It was not detected in both parents. A 2005 study by Bongers et al. described a significant association between the presence of clinically relevant renal involvement in an NPS patient and a positive family history of nephropathy, which was lacking in our case.