IDH2 inhibition enhances proteasome inhibitor responsiveness in hematological malignancies

Proteasome inhibitors (PIs) are extensively used for the therapy of multiple myeloma (MM) and mantle-cell lymphoma (MCL). However, patients continuously relapse or are intrinsically resistant to this class of drugs. Here, to identify targets that synergize with PIs, we carried out a functional screening in MM cell lines using a short hairpin RNA library against cancer driver genes. Isocitrate dehydrogenase 2 (IDH2) was identified as a top candidate, showing a synthetic lethal activity with the PI carfilzomib (CFZ). Combinations of FDA approved PIs with a pharmacological IDH2 inhibitor (AGI-6780) triggered synergistic cytotoxicity in MM, MCL, and Burkitt's lymphoma (BL) cell lines. CFZ/AGI-6780 treatment increased death of primary CD138+ cells from MM patients and exhibited a favorable cytotoxicity profile towards peripheral blood mononuclear cells and bone marrow-derived stromal cells. Mechanistically, CFZ/AGI-6780 combination significantly decreased tricarboxylic acid (TCA) cycle activity and ATP levels, as a consequence of enhanced IDH2 enzymatic inhibition. Specifically, CFZ treatment reduced the expression of nicotinamide phosphoribosyltransferase (NAMPT), thus limiting IDH2 activation through the NAD+-dependent deacetylase SIRT3. Consistently, combination of CFZ with either NAMPT or SIRT3 inhibitors impaired IDH2 activity and increased MM cell death. Finally, inducible IDH2 knockdown enhanced the therapeutic efficacy of CFZ in a subcutaneous xenograft model of MM, resulting in inhibition of tumor progression and extended survival. Taken together, these findings indicate that NAMPT/SIRT3/IDH2 pathway inhibition enhances the therapeutic efficacy of PIs, thus providing compelling evidence for treatments with lower and less toxic doses and broadening the application of PIs to other malignancies

Biomimetic mesoporous vectors enabling the efficient inhibition of wild-type isocitrate dehydrogenase in multiple myeloma cells

The discovery of isocitrate dehydrogenases (IDHs) mutations in several malignancies has brought to the approval of drugs targeting IDH1/2 mutants in cancers. More recently it has been suggested that the enzymatic inhibition of IDHs may have therapeutic potentials also for wild-type IDH cancers. Specifically, IDH2 inhibition can sensitize multiple myeloma cells to proteasome inhibitors. However, inhibitors directed against native IDHs are not present on the market. Here, we exploited an allosteric inhibitor of mutant IDH2 (AGI-6780), known to also decrease the activity of wild-type IDH2. Since AGI-6780 effectiveness in vivo is limited by its high hydrophobicity and very low bioavailability, the drug was loaded into mesoporous silica nanoparticles (MSNs) with the aim to enhance its efficacy. Furthermore, to enable high drug retention into the silica pores, improve biocompatibility, and reduce the off-target delivery of the drug, a Supported phosphoLipidic Bilayer (SLB) was self-assembled on the outer MSN surface. The silica nanoparticles were thus coated with three different lipid formulations and characterized in terms of structure, size, and morphology. We demonstrated that MSN@SLB nanoparticles have improved colloidal stability and hemocompatibility with respect to pristine MSN. We showed that MSN@SLB formulation displays an excellent loading and retention of the IDH2 inhibitor AGI-6780, with a limited drug leakage depending on the lipid formulation. Finally, we proved that AGI-6780-loaded MSN@SLB nanoparticles efficaciously inhibited the IDH2 enzymatic activity of multiple myeloma cells. Overall, this study provides a proof of concept of drug delivery to multiple myeloma cells by repurposing a neglected/dismissed drug (AGI-6780) with the use of smart nanoparticles and enabling the sensitization of multiple myeloma cells towards other possible treatments

Identification of a three-gene model as a powerful diagnostic tool for the recognition of ALK negative ALCL

Anaplastic Large Cell Lymphomas (ALCL) are a clinically and biologically heterogeneous disease including the ALK+ and ALK- systemic forms. While ALK+ ALCL are molecularly characterized and can be readily diagnosed, specific immunophenotypic or genetic features to define ALK- ALCL are missing, and their distinction from other T-Cell Non- Hodgkin Lymphomas (T-NHL) remains controversial. Here, we undertook a transcriptional profiling meta-analysis of 309 cases, including ALCL and other primary T-NHL samples. Pathway discovery and prediction analyses defined a minimum set of genes capable to recognize ALK- ALCL. Application of RT-qPCR in independent data sets from cryopreserved and formalin-fixed paraffin embedded (FFPE) samples validated a three-gene model (TNFRSF8, BATF3, TMOD1) able to successfully separate ALK- ALCL from PTCLNOS, with overall accuracy near 97%. In conclusion, our data justify the possibility to translate RT-qPCR protocols to routine clinical settings as a new approach to objectively dissect T-NHL and to select more appropriate therapeutic protocols

Biomimetic mesoporous vectors enabling the efficient inhibition of wild-type isocitrate dehydrogenase in multiple myeloma cells

The discovery of isocitrate dehydrogenases (IDHs) mutations in several malignancies has brought to the approval of drugs targeting IDH1/2 mutants in cancers. More recently it has been suggested that the enzymatic inhibition of IDHs may have therapeutic potentials also for wild-type IDH cancers. Specifically, IDH2 inhibition can sensitize multiple myeloma cells to proteasome inhibitors. However, inhibitors directed against native IDHs are not present on the market. Here, we exploited an allosteric inhibitor of mutant IDH2 (AGI-6780), known to also decrease the activity of wild-type IDH2. Since AGI-6780 effectiveness in vivo is limited by its high hydrophobicity and very low bioavailability, the drug was loaded into mesoporous silica nanoparticles (MSNs) with the aim to enhance its efficacy. Furthermore, to enable high drug retention into the silica pores, improve biocompatibility, and reduce the off-target delivery of the drug, a Supported phosphoLipidic Bilayer (SLB) was selfassembled on the outer MSN surface. The silica nanoparticles were thus coated with three different lipid formulations and characterized in terms of structure, size, and morphology. We demonstrated that MSN@SLB nanoparticles have improved colloidal stability and hemocompatibility with respect to pristine MSN. We showed that MSN@SLB formulation displays an excellent loading and retention of the IDH2 inhibitor AGI-6780, with a limited drug leakage depending on the lipid formulation. Finally, we proved that AGI-6780-loaded MSN@SLB nanoparticles efficaciously inhibited the IDH2 enzymatic activity of multiple myeloma cells. Overall, this study provides a proof of concept of drug delivery to multiple myeloma cells by repurposing a neglected/dismissed drug (AGI-6780) with the use of smart nanoparticles and enabling the sensitization of multiple myeloma cells towards other possible treatments

Identification of a 3-gene model as a powerful diagnostic tool for the recognition of ALK-negative anaplastic large-cell lymphoma

Anaplastic large-cell lymphomas (ALCLs) are a group of clinically and biologically heterogeneous diseases including the ALK(+) and ALK(-) systemic forms. Whereas ALK(+) ALCLs are molecularly characterized and can be readily diagnosed, specific immunophenotypic or genetic features to define ALK(-) ALCL are missing, and their distinction from other T-cell non-Hodgkin lymphomas (T-NHLs) remains controversial. In the present study, we undertook a transcriptional profiling meta-analysis of 309 cases, including ALCL and other primary T-NHL samples. Pathway discovery and prediction analyses defined a minimum set of genes capable of recognizing ALK(-) ALCL. Application of quantitative RT-PCR in independent datasets from cryopreserved and formalin-fixed paraffin-embedded samples validated a 3-gene model (TNFRSF8, BATF3, and TMOD1) able to successfully separate ALK(-) ALCL from peripheral T-cell lymphoma not otherwise specified, with overall accuracy near 97%. In conclusion, our data justify the possibility of translating quantitative RT-PCR protocols to routine clinical settings as a new approach to objectively dissect T-NHL and to select more appropriate therapeutic protocols

Identification of a new subclass of ALK negative ALCL expressing aberrant levels of ERBB4 transcripts

Anaplastic Large Cell Lymphoma (ALCL) is a clinical and biological heterogeneous disease including systemic ALK positive and ALK negative entities. To discover biomarkers and/or genes involved in ALK negative ALCL pathogenesis, we applied the Cancer Outlier Profile Analysis (COPA) algorithm to a gene expression profiling data set including 249 cases of T-cell non-Hodgkin lymphoma and normal T-cells. Ectopic co-expression of ERBB4 and COL29A1 genes was detected in 24% of ALK negative ALCL patients. RNA sequencing and 5'RNA Ligase-Mediated Rapid Amplification of cDNA Ends (RLM-RACE) identified two novel ERBB4 truncated transcripts, displaying intronic Transcription Start Sites. By luciferase assays we defined that the expression of ERBB4 aberrant transcripts is promoted by endogenous intronic Long Terminal Repeats (LTRs). ERBB4 expression was confirmed at protein level by western blotting and immunohistochemistry. Finally, we demonstrated that the expression of ERBB4 truncated forms show oncogenic potentials and that ERBB4 pharmacological inhibition partially controls ALCL cell growth and disease progression in an ERBB4 positive patient-derived tumorgraft model. In conclusion, we identified a new subclass of ALK negative ALCL characterized by aberrant expression of ERBB4 truncated transcripts carrying intronic 5'UTRs

Identification of a 3-gene model as a powerful diagnostic tool for the recognition of ALK-negative anaplastic large-cell lymphoma.

Anaplastic large-cell lymphomas (ALCLs) are a group of clinically and biologically heterogeneous diseases including the ALK(+) and ALK(-) systemic forms. Whereas ALK(+) ALCLs are molecularly characterized and can be readily diagnosed, specific immunophenotypic or genetic features to define ALK(-) ALCL are missing, and their distinction from other T-cell non-Hodgkin lymphomas (T-NHLs) remains controversial. In the present study, we undertook a transcriptional profiling meta-analysis of 309 cases, including ALCL and other primary T-NHL samples. Pathway discovery and prediction analyses defined a minimum set of genes capable of recognizing ALK(-) ALCL. Application of quantitative RT-PCR in independent datasets from cryopreserved and formalin-fixed paraffin-embedded samples validated a 3-gene model (TNFRSF8, BATF3, and TMOD1) able to successfully separate ALK(-) ALCL from peripheral T-cell lymphoma not otherwise specified, with overall accuracy near 97%. In conclusion, our data justify the possibility of translating quantitative RT-PCR protocols to routine clinical settings as a new approach to objectively dissect T-NHL and to select more appropriate therapeutic protocols