Prostate Cancer and the Mevalonate Pathway.

Antineoplastic therapies for prostate cancer (PCa) have traditionally centered around the androgen receptor (AR) pathway, which has demonstrated a significant role in oncogenesis. Nevertheless, it is becoming progressively apparent that therapeutic strategies must diversify their focus due to the emergence of resistance mechanisms that the tumor employs when subjected to monomolecular treatments. This review illustrates how the dysregulation of the lipid metabolic pathway constitutes a survival strategy adopted by tumors to evade eradication efforts. Integrating this aspect into oncological management could prove valuable in combating PCa.

Dynamic Changes in miRNA Expression during the Generation of Expanded and Activated NK Cells.

Therapies based on allogenic Natural Killer (NK) cells are becoming increasingly relevant, and our laboratory has produced expanded and activated NK (eNK) cells that are highly cytotoxic against several hematological cancers when used alone or in combination with currently approved therapeutic monoclonal antibodies. In order to produce eNK cells, healthy human donor NK cells undergo a 20-day expansion protocol with IL-2, IL-15 and Epstein-Barr virus (EBV)-transformed lymphoblastoid feeder cells. In order to produce an even more potent eNK-based therapy, we must elucidate the changes our protocol produces within healthy NK cells. To understand the post-transcriptional changes responsible for the increased cytolytic abilities of eNK cells, we performed microRNA (miRNA) expression analysis on purified NK cells from day 0 and day 20 of the protocol using quantitative reverse transcription PCR (RT-qPCR). Of the 384 miRNAs profiled, we observed changes in the expression of 64 miRNAs, with especially significant changes in 7 of them. The up-regulated miRNAs of note were miRs-146a, -124, -34a, and -10a, which are key in the regulation of cell survival through the modulation of pro-apoptotic genes such as PUMA. The down-regulation of miRs-199a, -223, and -340 was also detected and is associated with the promotion of NK cell cytotoxicity. We validated our analysis using immunoblot and flow cytometry studies on specific downstream targets of both up- and down-regulated miRNAs such as PUMA and Granzyme B. These results corroborate the functional importance of the described miRNA expression patterns and show the wide variety of changes that occur in eNK cells at day 20.

Independent genomic polymorphisms in the PknH serine threonine kinase locus during evolution of the Mycobacterium tuberculosis Complex affect virulence and host preference.

Species belonging to the Mycobacterium tuberculosis Complex (MTBC) show more than 99% genetic identity but exhibit distinct host preference and virulence. The molecular genetic changes that underly host specificity and infection phenotype within MTBC members have not been fully elucidated. Here, we analysed RD900 genomic region across MTBC members using whole genome sequences from 60 different MTBC strains so as to determine its role in the context of MTBC evolutionary history. The RD900 region comprises two homologous genes, pknH1 and pknH2, encoding a serine/threonine protein kinase PknH flanking the tbd2 gene. Our analysis revealed that RD900 has been independently lost in different MTBC lineages and different strains, resulting in the generation of a single pknH gene. Importantly, all the analysed M. bovis and M. caprae strains carry a conserved deletion within a proline rich-region of pknH, independent of the presence or absence of RD900. We hypothesized that deletion of pknH proline rich-region in M. bovis may affect PknH function, having a potential role in its virulence and evolutionary adaptation. To explore this hypothesis, we constructed two M. bovis 'knock-in' strains containing the M. tuberculosis pknH gene. Evaluation of their virulence phenotype in mice revealed a reduced virulence of both M. bovis knock-in strains compared to the wild type, suggesting that PknH plays an important role in the differential virulence phenotype of M. bovis vs M. tuberculosis.

Conjugation of the 9-kDa Isoform of Granulysin with Liposomes Potentiates Its Cytotoxicity.

Nine kDa granulysin (GRNLY) is a human cytolytic protein secreted by cytotoxic T lymphocytes (CTL) and NK cells of the immune system whose demonstrated physiological function is the elimination of bacteria and parasites. In previous studies by our group, the anti-tumor capacity of recombinant granulysin was demonstrated, both in vitro and in vivo. In the present work, we developed lipid nanoparticles whose surfaces can bind recombinant granulysin through the formation of a complex of coordination between the histidine tail of the protein and Ni2+ provided by a chelating lipid in the liposome composition and termed them LUV-GRNLY, for granulysin-bound large unilamellar vesicles. The objective of this formulation is to increase the granulysin concentration at the site of contact with the target cell and to increase the cytotoxicity of the administered dose. The results obtained in this work indicate that recombinant granulysin binds to the surface of the liposome with high efficiency and that its cytotoxicity is significantly increased when it is in association with liposomes. In addition, it has been demonstrated that the main mechanism of death induced by both granulysin and LUV-GRNLY is apoptosis. Jurkat-shBak cells are resistant to GRNLY and also to LUV-GRNLY, showing that LUV-GRNLY uses the mitochondrial apoptotic pathway to induce cell death. On the other hand, we show that LUV-GRNLY induces the expression of the pro-apoptotic members of the Bcl-2 family Bim and especially PUMA, although it also induced the expression of anti-apoptotic Bcl-xL. In conclusion, we demonstrate that binding of GRNLY to the surfaces of liposomes clearly augments its cytotoxic potential, with cell death executed mainly by the mitochondrial apoptotic pathway.

Immunologic evaluation and genetic defects of apoptosis in patients with autoimmune lymphoproliferative syndrome (ALPS).

Apoptosis plays an important role in controlling the adaptive immune response and general homeostasis of the immune cells, and impaired apoptosis in the immune system results in autoimmunity and immune dysregulation. In the last 25 years, inherited human diseases of the Fas-FasL pathway have been recognized. Autoimmune lymphoproliferative syndrome (ALPS) is an inborn error of immunity, characterized clinically by nonmalignant and noninfectious lymphoproliferation, autoimmunity, and increased risk of lymphoma due to a defect in lymphocyte apoptosis. The laboratory hallmarks of ALPS are an elevated percentage of T-cell receptor αß double negative T cells (DNTs), elevated levels of vitamin B12, soluble FasL, IL-10, IL-18 and IgG, and defective in vitro Fas-mediated apoptosis. In order of frequency, the genetic defects associated with ALPS are germinal and somatic ALPS-FAS, ALPS-FASLG, ALPS-CASP10, ALPS-FADD, and ALPS-CASP8. Partial disease penetrance and severity suggest the combination of germline and somatic FAS mutations as well as other risk factor genes. In this report, we summarize human defects of apoptosis leading to ALPS and defects that are known as ALPS-like syndromes that can be clinically similar to, but are genetically distinct from, ALPS. An efficient genetic and immunological diagnostic approach to patients suspected of having ALPS or ALPS-like syndromes is essential because this enables the establishment of specific therapeutic strategies for improving the prognosis and quality of life of patients.

Production of a Granulysin-Based, Tn-Targeted Cytolytic Immunotoxin Using Pulsed Electric Field Technology.

Granulysin is a protein present in the granules of human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, with cytolytic activity against microbes and tumors. Previous work demonstrated the therapeutic effect of the intratumoral injection of recombinant granulysin and of the systemic injection of an immunotoxin between granulysin and the anti-carcinoembryonic antigen single-chain Fv antibody fragment MFE23, which were produced in the yeast Pichia pastoris. In the present work, we developed a second immunotoxin combining granulysin and the anti-Tn antigen single-chain Fv antibody fragment SM3, that could have a broader application in tumor treatment than our previous immunotoxin. In addition, we optimized a method based on electroporation by pulsed electric field (PEF) to extract the remaining intracellular protein from yeast, augmenting the production and purificiation yield. The immunotoxin specifically recognized the Tn antigen on the cell surface. We also compared the thermal stability and the cytotoxic potential of the extracellular and intracellular immunotoxins on Tn-expressing human cell lines, showing that they were similar. Moreover, the bioactivity of both immunotoxins against several Tn+ cell lines was higher than that of granulysin alone.

Lipid Nanoparticles Decorated with TNF-Related Aptosis-Inducing Ligand (TRAIL) Are More Cytotoxic than Soluble Recombinant TRAIL in Sarcoma.

Sarcomas are rare and heterogeneous cancers classically associated with a poor outcome. Sarcomas are 1% of the cancer but recent estimations indicate that sarcomas account for 2% of the estimated cancer-related deaths. Traditional treatment with surgery, radiotherapy, and chemotherapy has improved the outcome for some types of sarcomas. However, novel therapeutic strategies to treat sarcomas are necessary. TNF-related apoptosis-inducing ligand (TRAIL) is a death ligand initially described as capable of inducing apoptosis on tumor cell while sparing normal cells. Only few clinical trials have used TRAIL-based treatments in sarcoma, but they show only low or moderate efficacy of TRAIL. Consequently, novel TRAIL formulations with an improved TRAIL bioactivity are necessary. Our group has developed a novel TRAIL formulation based on tethering this death ligand on a lipid nanoparticle surface (LUV-TRAIL) resembling the physiological secretion of TRAIL as a trasmembrane protein inserted into the membrane of exosomes. We have already demonstrated that LUV-TRAIL shows an improved cytotoxic activity when compared to soluble recombinant TRAIL both in hematological malignancies and epithelial-derived cancers. In the present study, we have tested LUV-TRAIL in several human sarcoma tumor cell lines with different sensitivity to soluble recombinant TRAIL, finding that LUV-TRAIL was more efficient than soluble recombinant TRAIL. Moreover, combined treatment of LUV-TRAIL with distinct drugs proved to be especially effective, sensitizing even more resistant cell lines to TRAIL.

TRAIL-coated lipid-nanoparticles overcome resistance to soluble recombinant TRAIL in non-small cell lung cancer cells.

PURPOSE: Non-small cell lung cancer (NSCLC) is one the types of cancer with higher prevalence and mortality. Apo2-Ligand/TRAIL is a TNF family member able to induce apoptosis in tumor cells but not in normal cells. It has been tested in clinical trials against different types of human cancer including NSCLC. However, results of clinical trials have shown a limited efficacy of TRAIL-based therapies. Recently we have demonstrated that artificial lipid nanoparticles coated with bioactive Apo2L/TRAIL (LUV-TRAIL) greatly improved TRAIL cytotoxic ability being capable of killing chemoresistant hematological cancer cells. In the present work we have extended the study to NSCLC. METHODS/PATIENTS: LUV-TRAIL-induced cytotoxicity was assessed on different NSCLC cell lines with different sensitivity to soluble TRAIL and on primary human tumor cells from three patients suffering from NSCLC cancer. We also tested LUV-TRAIL-cytotoxic ability in combination with several anti-tumor agents. RESULTS: LUV-TRAIL exhibited a greater cytotoxic effect compared to soluble TRAIL both in A549 cells and primary human NSCLC cells. LUV-TRAIL-induced cell death was dependent on caspase-8 and caspase-3 activation. Moreover, combination of LUV-TRAIL with other anti-tumor agents such as flavopiridol, and SNS-032 clearly enhanced LUV-TRAIL-induced cytotoxicity against NSCLC cancer cells. CONCLUSION: The novel formulation of TRAIL based on displaying it on the surface of lipid nanoparticles greatly increases its anti-tumor activity and has clinical potential in cancer treatment.

Decreased activation-induced cell death by EBV-transformed B-cells from a patient with autoimmune lymphoproliferative syndrome caused by a novel FASLG mutation.

BACKGROUND: Autoimmune lymphoproliferative syndrome (ALPS) is a primary immunodeficiency characterized by chronic lymphoproliferation, autoimmune manifestations, expansion of double-negative T-cells, and susceptibility to malignancies. Most cases of ALPS are caused by germline or somatic FAS mutations. We report the case of an ALPS patient due to a novel homozygous Fasligand gene mutation (ALPS-FASLG). METHODS: ALPS biomarkers were measured and FASLG mutation was identified. Functional characterization was carried out based on activation-induced cell death (AICD) and cytotoxicity assays. RESULTS: This report describes the cases of a patient who presented a severe form of ALPS-FASLG, and his brother who had died due to complications related to ALPS. Moreover, in another family, we present the first case of lymphoma in a patient with ALPS-FASLG. Functional studies showed defective Fasligand-mediated apoptosis, cytotoxicity, and AICD in T-cell blasts. Otherwise, expression of the FASLG gene and corresponding protein was normal, but the shedding of the Fasligand was impaired in T-cells. Additionally, analyzing Epstein-Barr virus (EBV)-transformed B-cells, our results indicate impaired AICD in ALPS-FASLG patients. CONCLUSION: Patients with autosomal recessive inheritance of ALPS-FASLG have a severe phenotype and a partial defect in AICD in T- and B-cell lines. The Fasligand could play a key role in immune surveillance preventing malignancy.

Regulation of PKD1-mediated Golgi to cell surface trafficking by Gαq subunits.

BACKGROUND INFORMATION: Heterotrimeric GTP-binding proteins play a key role in cell trafficking regulation. Above all, specific Gßγ subunits have been shown to be a major component of a signal transduction pathway, which also involves phospholipases C (PLC), protein kinases C (PKC) and D (PKD), whose main function is to regulate transport between Golgi and plasma membrane. It was the involvement of PLC which led us to study the role of the other member of this G protein family, the α subunits, in the regulation of membrane fission at the Golgi apparatus. RESULTS: Among constitutive active (QL) variants of different G protein α subunit sub-families, only GαqQL subunits were able to induce Golgi fragmentation, a phenotype that mainly reflects a membrane fission increase at this organelle. This phenotype was not observed with a GαqQL palmitoylation mutant, showing the need for a membrane-bounded subunit. Besides, GαqQL-dependent Golgi fission was blocked by specific PLC and PKC inhibitors, and in the presence of a PKD1-kinase dead variant. In addition, GαqQL was the only α subunit capable of inducing PKD1 phosphorylation. Finally, Vesicular Stomatitis Virus thermosensitive mutant glycoprotein (VSVG tsO45) transport assays have demonstrated that GαqQL acts directly on Golgi membranes to regulate trafficking between this organelle and plasma membrane. CONCLUSIONS: All these results indicate Gαq subunits for the first time as a regulator of PKD-mediated intracellular trafficking between Golgi apparatus and plasma membrane, opening new perspectives in the understanding of internal trafficking regulation by external signals through G protein-coupled receptors.

Liposomes decorated with Apo2L/TRAIL overcome chemoresistance of human hematologic tumor cells.

Human Apo2-ligand/TRAIL is a member of the TNF cytokine superfamily capable of inducing apoptosis on tumor cells while sparing normal cells. Besides its antitumor activity, Apo2L/TRAIL is also implicated in immune regulation. Apo2L/TRAIL is stored inside activated T cells in cytoplasmic multivesicular bodies and is physiologically released to the extracellular medium inserted in the internal membrane vesicles, known as exosomes. In this study we have generated artificial lipid vesicles coated with bioactive Apo2L/TRAIL, which resemble natural exosomes, to analyze their apoptosis-inducing ability on cell lines from hematological tumors. We have tethered Apo2L/TRAIL to lipid vesicles by using a novel Ni(2+)-(N-5-amino-1-carboxylpentyl)-iminodiacetic acid, NTA)-containing liposomal system. This lipidic framework (LUVs-Apo2L/TRAIL) greatly improves Apo2L/TRAIL activity, decreasing by around 14-fold the LC50 on the T-cell leukemia Jurkat. This increase in bioactivity correlated with the greater ability of LUVs-Apo2L/TRAIL to induce caspase-3 activation and is probably due to the increase in local concentration of Apo2L/TRAIL, improving its receptor cross-linking efficiency. More important, liposome-bound Apo2L/TRAIL overcame the resistance to soluble recombinant Apo2L/TRAIL exhibited by tumor cell mutants overexpressing Bcl-xL or by a Bax and Bak-defective Jurkat cell mutant (Jurkat-shBak) and are also effective against other hematologic tumor cells. Jurkat-Bcl-xL and Jurkat-shBak cells are resistant to most chemotherapeutic drugs currently used in cancer treatment, and their sensitivity to LUVs-Apo2L/TRAIL could have potential clinical applications.

Mitochondrial control of antigen presentation in cancer cells.

Cytotoxic T lymphocytes recognize and kill cancer cells when the latter present antigenic epitopes complexed with MHC class I molecules on their surface. In a recent Science paper, Mangalhara et al. show that alterations of the mitochondrial electron flow upregulate multiple factors involved in antigen presentation via a succinate-dependent epigenetic mechanism.

DR5 Up-Regulation Induced by Dichloroacetate Sensitizes Tumor Cells to Lipid Nanoparticles Decorated with TRAIL.

Cancer resistance to treatments is a challenge that researchers constantly seek to overcome. For instance, TNF-related apoptosis-inducing ligand (TRAIL) is a potential good prospect as an anti-cancer therapy, as it attacks tumor cells but not normal cells. However, treatments based in soluble TRAIL provided incomplete clinical results and diverse formulations have been developed to improve its bioactivity. In previous works, we generated a new TRAIL formulation based in its attachment to the surface of unilamellar nanoliposomes (LUV-TRAIL). This formulation greatly increased apoptosis in a wide selection of tumor cell types, albeit a few of them remained resistant. On the other hand, it has been described that a metabolic shift in cancer cells can also alter its sensitivity to other treatments. In this work, we sought to increase the sensitivity of several tumor cell types resistant to LUV-TRAIL by previous exposure to the metabolic drug dichloroacetate (DCA), which forces oxidative phosphorylation. Results showed that DCA + LUV-TRAIL had a synergistic effect on both lung adenocarcinoma A549, colorectal HT29, and breast cancer MCF7 cells. Despite DCA inducing intracellular changes in a cell-type specific way, the increase in cell death by apoptosis was clearly correlated with an increase in death receptor 5 (DR5) surface expression in all cell lines. Therefore, DCA-induced metabolic shift emerges as a suitable option to overcome TRAIL resistance in cancer cells.

Study of the Bcl-2 Interactome by BiFC Reveals Differences in the Activation Mechanism of Bax and Bak.

Evasion of apoptosis is one of the hallmarks of cancer cells. Proteins of the Bcl-2 family are key regulators of the intrinsic pathway of apoptosis, and alterations in some of these proteins are frequently found in cancer cells. Permeabilization of the outer mitochondrial membrane, regulated by pro- and antiapoptotic members of the Bcl-2 family of proteins, is essential for the release of apoptogenic factors leading to caspase activation, cell dismantlement, and death. Mitochondrial permeabilization depends on the formation of oligomers of the effector proteins Bax and Bak after an activation event mediated by BH3-only proteins and regulated by antiapoptotic members of the Bcl-2 family. In the present work, we have studied interactions between different members of the Bcl-2 family in living cells via the BiFC technique. Despite the limitations of this technique, present data suggest that native proteins of the Bcl-2 family acting inside living cells establish a complex network of interactions, which would fit nicely into "mixed" models recently proposed by others. Furthermore, our results point to differences in the regulation of Bax and Bak activation by proteins of the antiapoptotic and BH3-only subfamilies. We have also applied the BiFC technique to explore the different molecular models proposed for Bax and Bak oligomerization. Bax and Bak's mutants lacking the BH3 domain were still able to associate and give BiFC signals, suggesting the existence of alternative surfaces of interaction between two Bax or Bak molecules. These results agree with the widely accepted symmetric model for the dimerization of these proteins and also suggest that other regions, different from the α6 helix, could be involved in the oligomerization of BH3-in groove dimers.

Transmitochondrial Cybrid Generation Using Cancer Cell Lines.

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more efforts are still needed to fully understand the link involving alterations in mitochondria and tumorigenesis, as well as to identify tumor-associated mitochondrial phenotypes. For instance, to evaluate the contribution of mitochondria in tumorigenesis and metastasis processes, it is essential to understand the influence of mitochondria from tumor cells in different nuclear environments. For this purpose, one possible approach consists of transferring mitochondria into a different nuclear background to obtain the so-called cybrid cells. In the traditional cybridization techniques, a cell line lacking mtDNA (ρ0, nuclear donor cell) is repopulated with mitochondria derived from either enucleated cells or platelets. However, the enucleation process requires good cell adhesion to the culture plate, a feature that is partially or completely lost in many cases in invasive cells. In addition, another difficulty found in the traditional methods is achieving complete removal of the endogenous mtDNA from the mitochondrial-recipient cell line to obtain pure nuclear and mitochondrial DNA backgrounds, avoiding the presence of two different mtDNA species in the generated cybrid. In this work, we present a mitochondrial exchange protocol applied to suspension-growing cancer cells based on the repopulation of rhodamine 6G-pretreated cells with isolated mitochondria. This methodology allows us to overcome the limitations of the traditional approaches, and thus can be used as a tool to expand the comprehension of the mitochondrial role in cancer progression and metastasis.

Dinaciclib synergizes with BH3 mimetics targeting BCL-2 and BCL-XL in multiple myeloma cell lines partially dependent on MCL-1 and in plasma cells from patients.

A better understanding of multiple myeloma (MM) biology has led to the development of novel therapies. However, MM is still an incurable disease and new pharmacological strategies are needed. Dinaciclib, a multiple cyclin-dependent kinase (CDK) inhibitor, which inhibits CDK1, 2, 5 and 9, displays significant antimyeloma activity as found in phase II clinical trials. In this study, we have explored the mechanism of dinaciclib-induced death and evaluated its enhancement by different BH3 mimetics in MM cell lines as well as in plasma cells from MM patients. Our results indicate a synergistic effect of dinaciclib-based combinations with B-cell lymphoma 2 or B-cell lymphoma extra-large inhibitors, especially in MM cell lines with partial dependence on myeloid cell leukemia sequence 1 (MCL-1). Simultaneous treatment with dinaciclib and BH3 mimetics ABT-199 or A-1155463 additionally showed a synergistic effect in plasma cells from MM patients, ex vivo. Altered MM cytogenetics did not affect dinaciclib response ex vivo, alone or in combined treatment, suggesting that these combinations could be a suitable therapeutic option for patients bearing cytogenetic alterations and poor prognosis. This work also opens the possibility to explore cyclin-dependent kinase 9 inhibition as a targeted therapy in MM patients overexpressing or with high dependence on MCL-1.

Oxidative phosphorylation induces de novo expression of the MHC class I in tumor cells through the ERK5 pathway.

Most cancer cells use anaerobic-like glycolysis to generate energy instead of oxidative phosphorylation. They also avoid recognition by CTLs, which occurs primarily through decreasing the level of MHC class I (MHC-I) at the cell surface. We find that the two phenomena are linked; culture conditions that force respiration in leukemia cells upregulate MHC-I transcription and protein levels at the cell surface, whereas these decrease in cells forced to perform fermentation as well as in leukemia cells lacking a functional mitochondrial respiratory chain. Forced respiration leads to increased expression of the MAPK ERK5, which activates MHC-I gene promoters, and ERK5 accumulation in mitochondria. Respiration-induced MHC-I upregulation is reversed upon short hairpin RNA-mediated ERK5 downregulation and by inactive mutants of ERK5. Moreover, short hairpin RNA for ERK5 leukemia cells do not tolerate forced respiration. Thus, the expression of ERK5 and MHC-I is linked to cell metabolism and notably diminished by the metabolic adaptations found in tumor cells.

Harnessing the Potential of NK Cell-Based Immunotherapies against Multiple Myeloma.

Natural killer (NK) cell-based therapies have emerged as promising anticancer treatments due to their potency as cytolytic effectors and synergy with concurrent treatments. Multiple myeloma (MM) is an aggressive B-cell malignancy that, despite development of novel therapeutic agents, remains incurable with a high rate of relapse. In MM, the inhospitable tumor microenvironment prevents host NK cells from exerting their cytolytic function. The development of NK cell immunotherapy works to overcome this altered immune landscape and can be classified in two major groups based on the origin of the cell: autologous or allogeneic. In this review, we compare the treatments in each group, such as autologous chimeric antigen receptor (CAR) NKs and allogeneic off-the-shelf NK cell infusions, and their combinatorial effect with existing MM therapies including monoclonal antibodies and proteasome inhibitors. We also discuss their placement in clinical treatment regimens based on the immune profile of each patient. Through this examination, we would like to discover precisely when each NK cell-based treatment will produce the maximum benefit to the MM patient.

Ecto-calreticulin expression in multiple myeloma correlates with a failed anti-tumoral immune response and bad prognosis.

Immunogenic cell death (ICD) has been proposed to be a crucial process for antitumor immunosurveillance. ICD is characterized by the exposure and emission of Damage Associated Molecular Patterns (DAMP), including calreticulin (CRT). A positive correlation between CRT exposure or total expression and improved anticancer immunosurveillance has been found in certain cancers, usually accompanied by favorable patient prognosis. In the present study, we sought to evaluate CRT levels in the plasma membrane of CD38+ bone marrow mononuclear cells (BMMCs) isolated from 71 patients with varying degrees of multiple myeloma (MM) disease and examine the possible relationship between basal CRT exposure and the bone marrow immune microenvironment, as well as its connection with different clinical markers. Data show that increased levels of cell surface-CRT were associated with more aggressive clinical features and with worse clinical prognosis in MM. High CRT expression in MM cells was associated with increased infiltration of NK cells, CD8+ T lymphocytes and dendritic cells (DC), indicative of an active anti-tumoral immune response, but also with a significantly higher presence of immunosuppressive Treg cells and increased expression of PD-L1 in myeloma cells.

PT-112 Induces Mitochondrial Stress and Immunogenic Cell Death, Targeting Tumor Cells with Mitochondrial Deficiencies.

PT-112 is a novel pyrophosphate-platinum conjugate, with clinical activity reported in advanced pretreated solid tumors. While PT-112 has been shown to induce robust immunogenic cell death (ICD) in vivo but only minimally bind DNA, the molecular mechanism underlying PT-112 target disruption in cancer cells is still under elucidation. The murine L929 in vitro system was used to test whether differential metabolic status alters PT-112's effects, including cell cytotoxicity. The results showed that tumor cells presenting mutations in mitochondrial DNA (mtDNA) (L929dt and L929dt cybrid cells) and reliant on glycolysis for survival were more sensitive to cell death induced by PT-112 compared to the parental and cybrid cells with an intact oxidative phosphorylation (OXPHOS) pathway (L929 and dtL929 cybrid cells). The type of cell death induced by PT-112 did not follow the classical apoptotic pathway: the general caspase inhibitor Z-VAD-fmk did not inhibit PT-112-induced cell death, alone or in combination with the necroptosis inhibitor necrostatin-1. Interestingly, PT-112 initiated autophagy in all cell lines, though this process was not complete. Autophagy is known to be associated with an integrated stress response in cancer cells and with subsequent ICD. PT-112 also induced a massive accumulation of mitochondrial reactive oxygen species, as well as changes in mitochondrial polarization-only in the sensitive cells harboring mitochondrial dysfunction-along with calreticulin cell-surface exposure consistent with ICD. PT-112 substantially reduced the amount of mitochondrial CoQ10 in L929 cells, while the basal CoQ10 levels were below our detection limits in L929dt cells, suggesting a potential relationship between a low basal level of CoQ10 and PT-112 sensitivity. Finally, the expression of HIF-1α was much higher in cells sensitive to PT-112 compared to cells with an intact OXPHOS pathway, suggesting potential clinical applications.