A drug repurposing strategy for overcoming human multiple myeloma resistance to standard-of-care treatment

Despite several approved therapeutic modalities, multiple myeloma (MM) remains an incurable blood malignancy and only a small fraction of patients achieves prolonged disease control. The common anti-MM treatment targets proteasome with specific inhibitors (PI). The resulting interference with protein degradation is particularly toxic to MM cells as they typically accumulate large amounts of toxic proteins. However, MM cells often acquire resistance to PIs through aberrant expression or mutations of proteasome subunits such as PSMB5, resulting in disease recurrence and further treatment failure. Here we propose CuET—a proteasome-like inhibitor agent that is spontaneously formed in-vivo and in-vitro from the approved alcohol-abuse drug disulfiram (DSF), as a readily available treatment effective against diverse resistant forms of MM. We show that CuET efficiently kills also resistant MM cells adapted to proliferate under exposure to common anti-myeloma drugs such as bortezomib and carfilzomib used as the first-line therapy, as well as to other experimental drugs targeting protein degradation upstream of the proteasome. Furthermore, CuET can overcome also the adaptation mechanism based on reduced proteasome load, another clinically relevant form of treatment resistance. Data obtained from experimental treatment-resistant cellular models of human MM are further corroborated using rather unique advanced cytotoxicity experiments on myeloma and normal blood cells obtained from fresh patient biopsies including newly diagnosed as well as relapsed and treatment-resistant MM. Overall our findings suggest that disulfiram repurposing particularly if combined with copper supplementation may offer a promising and readily available treatment option for patients suffering from relapsed and/or therapy-resistant multiple myeloma.Subject terms: Myeloma, Cancer therapeutic resistance     

A potent inhibitor of cell proliferation in "middle molecules" isolated from the urine of uremic patients

A potent inhibitor of cell proliferation was found in the urine of a patient with chronic renal failure. This substance included in "middle molecules" (MM) fraction, was obtained by chromatography. This factor was shown to inhibit noticeably the proliferation of various cells : lymphocytes stimulated by allogeneic cells, monolayer cell lines of normal or tumorous origin and leukemic cell lines derived from acute lymphoblastic leukemia. This effect was reversible and thus could not be related to a direct, rapid cytotoxic effect of MM. Such substances could play an important part in uremic symptoms, such as immunodeficiency, anemia, thrombopenia, gastrointestinal or skin manifestations.     

Impaired germ cell development due to compromised cell cycle progression in Skp2-deficient mice

Background

A high proliferative capacity of tumor cells usually is associated with shortened patient survival. Disruption of the RB pathway, which is critically involved in regulating the G1 to S cell cycle transition, is a frequent target of oncogenic events that are thought to contribute to increased proliferation during tumor progression. Previously, we determined that p18INK4c, an essential gene for normal plasma cell differentiation, was bi-allelically deleted in five of sixteen multiple myeloma (MM) cell lines. The present study was undertaken to investigate a possible role of p18INK4c in increased proliferation of myeloma tumors as they progress.

Results

Thirteen of 40 (33%) human myeloma cell lines do not express normal p18INK4c, with bi-allelic deletion of p18 in twelve, and expression of a mutated p18 fragment in one. Bi-allelic deletion of p18, which appears to be a late progression event, has a prevalence of about 2% in 261 multiple myeloma (MM) tumors, but the prevalence is 6 to10% in the 50 tumors with a high expression-based proliferation index. Paradoxically, 24 of 40 (60%) MM cell lines, and 30 of 50 (60%) MM tumors with a high proliferation index express an increased level of p18 RNA compared to normal bone marrow plasma cells, whereas this occurs in only five of the 151 (3%) MM tumors with a low proliferation index. Tumor progression is often accompanied by increased p18 expression and an increased proliferation index. Retroviral-mediated expression of exogenous p18 results in marked growth inhibition in three MM cell lines that express little or no endogenous p18, but has no effect in another MM cell line that already expresses a high level of p18.

Conclusion

Paradoxically, although loss of p18 appears to contribute to increased proliferation of nearly 10% of MM tumors, most MM cell lines and proliferative MM tumors have increased expression of p18. Apart from a small fraction of cell lines and tumors that have inactivated the RB1 protein, it is not yet clear how other MM cell lines and tumors have become insensitive to the anti-proliferative effects of increased p18 expression.     

Molecular mechanics (MM4) force field development for phosphine and its alkyl derivatives

Based on the experimental structures and vibrational spectra of eight alkylphosphines, as well as ten ab initio calculated (MP2/6-31+g*) structures and five potential energy profiles, the MM4 force field has been extended to include this important functional group. The results are comparable to experimental values for phosphorus-containing compounds. The addition of various cross-terms significantly improved the MM4 calculated structures relative to its predecessor, MM3. The overall root mean square error in moments of inertia have been reduced from 1.65% in MM3 to 0.26% in MM4. MM4 was also successful in reducing the previously impressive root mean square error in vibrational frequencies from 35 cm(-1) to 31 cm(-1) for the eight compounds studied.     

The differential modulation of the enzymes of glutathione metabolism

The effect of methylmercury (MM) and MM plus sodium selenite (SE) on the activity of various GSH-dependent enzymes was studied in the liver and kidney of mice. Ten groups of mice were fed diets containing graded proportions of MM, alone or with graded quantities of SE. GST, GSH-Px, and GSSG-RED were assayed in the cytosolic fraction of liver and kidney homogenates. After treatment with MM, instead of the expected decrease in enzyme activities, an increase was observed in the kidney and a small decrease in the liver with no dose-response relation in either organ. In protected groups, a general pattern of induction was observed in both organs, but again there was little evidence of dose-response relationships. Detailed analysis of the results suggests that the effects observed were not directly caused by MM or SE but are the resultant of complex interactions presumably related to contemporaneous mechanisms of damage and repair.     

Cytotoxicities of two disulfide-bond-linked conjugates of methotrexate with monoclonal anti-MM46 antibody

Summary In studies on (antitumor antibody)-drug conjugates as potential antitumor agents, the amide derivatives of methotrexate (MTX) with cysteine and with 2-mercaptoethylamine (cysteamine) (MTX-Cys and MTX-MEA, respectively) were linked via a disulfide bond with a monoclonal antibody (MM46) to a mouse mammary tumor MM46 with attached 3-(2-pyridyldithio) propionyl groups to give conjugates of MTX with MM46 (MTX-Cys-SS-MM46 and MTX-MEA-SS-MM46, respectively). These two conjugates are both linked by a disulfide bond and are very similar in structure, but MTX-MEA-SS-MM46 showed only weak in vitro cytotoxicity against MM46 cells, whereas MTX-Cys-SS-MM46 had strong cytotoxicity. The cytotoxicity of the latter was comparable to that of the conventional direct MTX-MM46 conjugate prepared with an MTX-active ester. However, this conjugate had a greater selectivity than that of the direct conjugate, calculated as the IC₅₀ (concentration of a conjugate by MTX equivalence required for suppression of the number of viable MM46 cells to 50% of that of the untreated control) for the corresponding nonspecific conjugate divided by the IC₅₀ for the MM46 conjugate. The inhibitory activities of MTX-Cys and MTX-MEA on dihydrofolate reductase were similar. The cytotoxicity of MTX-Cys-SS-MM46 was not affected by thiamine pyrophosphate, an inhibitor of the active transport of MTX across the cell membrane, but was decreased significantly by ammonium chloride, a lysosomotropic amine. However, the cytotoxicity was decreased only to a small extent by leupeptin, an inhibitor of lysosomal cysteine proteases cathepsins B, H, and L. These results suggest that the cytotoxicity is mediated by lysosomes, and may involve lysosomal enzymes other than cathepsins B, H, and L.     

The use of MM3 monoclonal antibodies for the early immunodiagnosis of ovine fascioliasis

This study reports a new capture ELISA (MM3-SERO) for the serodiagnosis of sheep fascioliasis, based on the use of the monoclonal antibody (mAb) MM3. Like our previously reported indirect ELISA method, based on the use of a FPLC-purified fraction (fraction IV) of the Fasciola hepatica excretion/secretion antigens (ESAs), this new test was able to detect animals infected with very small numbers of metacercariae (5-40) and showed no cross-reaction with sera from sheep infected with other parasites, i.e., Moniezia spp., Cysticercus tenuicollis, and Dicrocoelium dendriticum. In contrast with these 2 methods, some sera (mainly those obtained from animals infected with D. dendriticum) showed high reactivities in indirect ELISA with whole F. hepatica ESAs used as control. Interestingly, the MM3-SERO ELISA has a better signal-to-noise ratio than the fraction-IV ELISA, thus allowing detection of seroconversion in infected sheep on average 1 wk earlier (3.2 +/- 0.4 wk postinfection [PI] for MM3-SERO ELISA vs. 4.2 +/- 0.9 wk PI for fraction IV ELISA). Moreover, the antibody response detected with MM3-SERO ELISA was more uniform, with seroconversion always occurring at 4 wk PI in sheep with 1-2 flukes and at 3 wk PI in sheep with more than 2 flukes. The MM3-SERO ELISA was also used to evaluate the kinetics of antibody response against MM3-recognized antigens in sera from sheep experimentally infected with F. hepatica and then treated with triclabendazole. Our results showed that antibody levels dropped by about 25% during the 4-wk observation period following the flukicide treatment, whereas they remained invariably high in all sheep left untreated. We conclude that the MM3-SERO ELISA is a 100% sensitive and 100% specific test for the early serodiagnosis of sheep fascioliasis. Preliminary studies in our laboratory seem to indicate that this method may also be useful for the determination of anti-F. hepatica antibodies in serum and milk of other ruminants. A commercial version of MM3-SERO is currently available from BIO X Diagnostics (La Jemelle, Belgium).     

Malignant melanoma in Chile: different site distribution between private and state patients

Background

The body site location of primary Malignant Melanoma (MM) has been correlated with prognosis and survival. Ethnic, genetics, sun exposure factors are related to the anatomical distribution of MM. Low and high socioeconomic strata in Chile differ in ethnic, genetic and cultural conditions. The purpose of this study was to analyze the anatomical MM distribution in the Chilean population in both strata searching for differences due to their ethno-genetic-cultural differences. Records of 1148 MM, 575cases from state hospitals (Low Socioeconomic Strata, LSS) and 573 cases from private clinics (High Socioeconomic Strata, HSS) were analyzed by body site.

Results

Females from LSS showed a higher number of MM in soles, cheeks, and around the eye area. Females from the HSS showed a higher number of MM in dorsal feet and dorsal hands. Males from LSS showed a higher number of MM in soles, around the eye area, and cheeks. However, males from HSS showed a higher number of MM in the trunk, and in the arms. Acral MM was significantly higher in LSS than in the HSS in both sexes. The Chilean population from the HSS and LSS showed differences in the distribution of MM by site. Furthermore, gender differences in the proportion of MM analyzed by anatomical site are observed in both strata.

Conclusions

Results show evidence that differential genetics factors, sun exposure, or other environmental or cultural factors of both strata may account for these differences.     

Role of Wnt5a-Ror2 Signaling in Morphogenesis of the Metanephric Mesenchyme during Ureteric Budding

Development of the metanephric kidney begins with the induction of a single ureteric bud (UB) on the caudal Wolffian duct (WD) in response to GDNF (glial cell line-derived neurotrophic factor) produced by the adjacent metanephric mesenchyme (MM). Mutual interaction between the UB and MM maintains expression of GDNF in the MM, thereby supporting further outgrowth and branching morphogenesis of the UB, while the MM also grows and aggregates around the branched tips of the UB. Ror2, a member of the Ror family of receptor tyrosine kinases, has been shown to act as a receptor for Wnt5a to mediate noncanonical Wnt signaling. We show that Ror2 is predominantly expressed in the MM during UB induction and that Ror2- and Wnt5a-deficient mice exhibit duplicated ureters and kidneys due to ectopic UB induction. During initial UB formation, these mutant embryos show dysregulated positioning of the MM, resulting in spatiotemporally aberrant interaction between the MM and WD, which provides the WD with inappropriate GDNF signaling. Furthermore, the numbers of proliferating cells in the mutant MM are markedly reduced compared to the wild-type MM. These results indicate an important role of Wnt5a-Ror2 signaling in morphogenesis of the MM to ensure proper epithelial tubular formation of the UB required for kidney development.     

Search for peptidic "middle molecules" in uremic sera: isolation and chemical identification of fibrinogen fragments

According to the "middle molecule" (MM) hypothesis, the uremic solutes ranging from 500 to 5,000 Da are insufficiently eliminated by conventional hemodialysis and may act as uremic toxins. However, because of the methodological difficulties of MM purification, their chemical analysis is complicated and the precise structure of these molecules remains obscure. In the present study, a new micro-preparative procedure including SDS electrophoresis and liquid chromatography was applied for isolation of MM peptides from uremic sera. Microsequencing and MS/MS analyses of these peptides showed that most of the identified MM (22 out of 23) represented the N- and C-terminal fragments of the alpha- and beta-chains of fibrinogen. The obtained data provide new information on the precise structure of fibrinogen fragments accumulating in uremic serum as MM.     

Transmissible cytotoxicity of multiple myeloma cells by cord blood-derived NK cells is mediated by vesicle trafficking

Natural killer cells (NK) are important effectors of anti-tumor immunity, activated either by the downregulation of HLA-I molecules on tumor cells and/or the interaction of NK-activating receptors with ligands that are overexpressed on target cells upon tumor transformation (including NKG2D and NKP30). NK kill target cells by the vesicular delivery of cytolytic molecules such as Granzyme-B and Granulysin activating different cell death pathways, which can be Caspase-3 dependent or Caspase-3 independent. Multiple myeloma (MM) remains an incurable neoplastic plasma-cell disorder. However, we previously reported the encouraging observation that cord blood-derived NK (CB-NK), a new source of NK, showed anti-tumor activity in an in vivo murine model of MM and confirmed a correlation between high levels of NKG2D expression by MM cells and increased efficacy of CB-NK in reducing tumor burden. We aimed to characterize the mechanism of CB-NK-mediated cytotoxicity against MM cells. We show a Caspase-3- and Granzyme-B-independent cell death, and we reveal a mechanism of transmissible cell death between cells, which involves lipid–protein vesicle transfer from CB-NK to MM cells. These vesicles are secondarily transferred from recipient MM cells to neighboring MM cells amplifying the initial CB-NK cytotoxicity achieved. This indirect cytotoxicity involves the transfer of NKG2D and NKP30 and leads to lysosomal cell death and decreased levels of reactive oxygen species in MM cells. These findings suggest a novel and unique mechanism of CB-NK cytotoxicity against MM cells and highlight the importance of lipids and lipid transfer in this process. Further, these data provide a rationale for the development of CB-NK-based cellular therapies in the treatment of MM.Natural killer cells (NK) are important effectors of anti-tumor immunity of the immune system. They can be activated by inhibition of killer cell immunoglobulin (Ig)-like receptor (KIR) receptors on NK due to downregulation of HLA-I on tumor cells or by the interaction of NK-activating receptors with ligands that are overexpressed on target cells. These receptors include NKG2D and the family of NK cytotoxicity receptors (NKP30, NKP44, NKP46),¹ which can be on the cell surface and in the endocytic compartment² from where they traffic to tumor cells in exosomes to exert cytotoxicity.³NK deliver cytolytic molecules such as Granzyme-B (GrB) and Granulysin to target cells. GrB induces Caspase-3-dependent apoptotic cell death with reactive oxygen species (ROS) generation.⁴ Alternatively, Granulysin activates Caspase-3-dependent cell death through ROS generation^{5, 6} and Caspase-3-independent cell death via endoplasmic reticulum (ER) stress⁷ or lysosomal cell death through release of cathepsins.⁸Multiple myeloma (MM) is a plasma-cell disorder characterized by clonal proliferation of malignant plasma-cells in the bone marrow (BM) and monoclonal protein in the blood or urine.^{9, 10} Plasma cells synthesize large quantities of Igs, which are folded in the ER. An excess of Ig synthesis causes a failure in this folding process leading to the release of unfolded/misfolded Igs.¹¹ These Igs are degraded by intracellular protein degradation pathways, including the ubiquitin–proteasome system and autophagy. Proteasome inhibitors (PIs) are potent anti-MM agents,¹² which block the protein degradation process in MM cells leading to ER stress-mediated cell death.^{13, 14} An excess of PI-induced ER stress can increase autophagy^{15, 16} leading to eventual refractory disease in some patients.^{17, 18, 19} Therefore new anti-MM strategies are needed.Previously, we have demonstrated that cord blood-derived NK (CB-NK) show anti-tumor activity in an in vivo MM murine model²⁰ and observed that the expression of NKG2D by MM tumor cells correlated with lower tumor burden following CB-NK cellular therapy. Here we characterize the CB-NK-mediated cytotoxicity against MM cells and observe a Caspase-3- and Gr-B-independent cell death and reveal a mechanism of transmissible cell death between cells that involves lipid–protein vesicle transfer from CB-NK to MM cells. These vesicles are secondarily transferred from recipient MM cells to neighboring MM cells, thereby amplifying the initial CB-NK cytotoxicity achieved. This indirect cytotoxicity involves the transfer of NKG2D and NKP30 and leads to lysosomal cell death and reduced ROS levels in MM cells.     

Physical parameter estimation from porcine ex vivo vocal fold dynamics in an inverse problem framework

This study presents a framework for a direct comparison of experimental vocal fold dynamics data to a numerical two-mass-model (2MM) by solving the corresponding inverse problem of which parameters lead to similar model behavior. The introduced 2MM features improvements such as a variable stiffness and a modified collision force. A set of physiologically sensible degrees of freedom is presented, and three optimization algorithms are compared on synthetic vocal fold trajectories. Finally, a total of 288 high-speed video recordings of six excised porcine larynges were optimized to validate the proposed framework. Particular focus lay on the subglottal pressure, as the experimental subglottal pressure is directly comparable to the model subglottal pressure. Fundamental frequency, amplitude and objective function values were also investigated. The employed 2MM is able to replicate the behavior of the porcine vocal folds very well. The model trajectories’ fundamental frequency matches the one of the experimental trajectories in \(98.6\%\) of the recordings. The relative error of the model trajectory amplitudes is on average \(9.5\%\). The experiments feature a mean subglottal pressure of 10.16 (SD \(= 2.31\)) \({\text {cmH}}_2{\text {O}}\); in the model, it was on average 7.61 (SD \(= 2.40\)) \({\text {cmH}}_2{\text {O}}\). A tendency of the model to underestimate the subglottal pressure is found, but the model is capable of inferring trends in the subglottal pressure. The average absolute error between the subglottal pressure in the model and the experiment is 2.90 (SD \(= 1.80\)) \({\text {cmH}}_2{\text {O}}\) or \(27.5\%\). A detailed analysis of the factors affecting the accuracy in matching the subglottal pressure is presented.     

High‐definition optical coherence tomography of melanocytic skin lesions

High‐definition optical coherence tomography (HD‐OCT) scanners have recently been developed. We assessed micromorphological HD‐OCT correlates of benign naevi (BN) and malignant melanoma (MM). 28 BN and 20 MM were studied using HD‐OCT and histology. Epidermal honeycomb/cobblestone pattern, regular junctional cell nests, and edged papillae are more often observed in BN, whereas fusion of rete ridges, pagetoid cells and junctional and/or dermal nests with atypical cells are more frequently seen in MM. A high overlap of HD‐OCT features in BN and MM was observed and in 20% of MM we did not find evidence for malignancy in OCT images at all. Using HD‐OCT it is possible to visualize architectural and cellular alterations of melanocytic skin lesions. The overlap of HD‐OCT features seen in BN and MM and the absence of suspicious HD‐OCT features in some MM represents an important limitation of HD‐OCT affecting the sensitivity of HD‐OCT in diagnosing MM.

High‐definition optical coherence tomography and the corresponding vertically sectioned histology of a compound naevus.     

Exosome-transmitted LINC00461 promotes multiple myeloma cell proliferation and suppresses apoptosis by modulating microRNA/BCL-2 expression

Background

Multiple myeloma (MM) is a hematologic cancer caused by the abnormal expansion of plasma cells, but the exact mechanism underlying MM development is not completely known. Recently, multiple long noncoding RNAs (lncRNAs) were implicated in the regulation of MM development.

Creatine kinase of rat lymphocytes and its activity as affected by adenosine, cAMP, AMP and products of their transformation

Creatine kinase is found in the thymus and spleen lymphocytes of rats. Its activity in the spleen lymphocytes is considerably higher than in the thymocytes. The data of enzyme-electrophoresis chromatography on DEAE-Sephadex A-50, thermostability and pH optimum indicate that lymphocyte creatine kinase is a "cerebral" isoenzyme. It is mainly concentrated in hyaloplasm and mitochondrial fraction and, to a less extent--in the microsomal and nuclear-cellular fractions. It is shown that adenosine, inosine, hypoxanthin, NH4, cAMP and GTP have no effect on the creatine kinase activity of thymocytes but pyrophosphate causes its decrease. Creatine kinase of lymphocytes as compared with its myocardium isoenzyme MM possesses a less substrate specificity to nucleotide diphosphates.     

Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry

Background

In order to provide reliable tissue material for malignant mesothelioma (MM) studies, we re-evaluated biopsies and autopsy material from 61 patients with a diagnosis of MM from the period of 1980-2002.

Methods

Basic positive (Calretinin, EMA, Podoplanin, Mesothelin) and negative (CEA, Ber-Ep4) immunohistochemical (IHC) marker reactions were determined. If needed, more markers were used. Histological diagnoses were made by three pathologists. Survival data were calculated.

Results

49 cases (80%) were considered being MM by a high degree of likelihood, five more cases possible MM. Of the remaining seven cases, three were diagnosed as adenocarcinoma, three as pleomorphic lung carcinoma, in one peritoneal case a clear entity diagnosis could not be given. One of the possible MM cases and two of the lung carcinoma cases had this already as primary diagnoses, but were registered as MM. With a sensitivity of 100%, Calretinin and CEA were the most reliable single markers. The amount of MM cells with positive immunoreactivity (IR) for Podoplanin and Mesothelin showed most reliable inverse relation to the degree of atypia. In the confirmed MM cases, there had been applied either no IHC or between one and 18 markers. The cases not confirmed by us had either lacked IHC (n = 1), non-specific markers were used (n = 4), IR was different (n = 1), or specific markers had not shown positive IR in the right part of the tumour cells (n = 3). 46 of the 49 confirmed and three of the not confirmed cases had been diagnosed by us as most likely MM before IHC was carried out.

Conclusions

In order to use archival tissue material with an earlier MM diagnosis for studies, histopathological re-evaluation is important. In possible sarcomatous MM cases without any positive IR for positive MM markers, radiology and clinical picture are essential parts of diagnostics. IHC based on a panel of two positive and two negative MM markers has to be adapted to the differential diagnostic needs in each single case. New diagnostic tools and techniques are desirable for cases where IHC and other established methods cannot provide a clear entity diagnosis, and in order to improve MM treatment.