Cereblon enhancer methylation and IMiD resistance in multiple myeloma.

Cereblon is the direct binding target of the immunomodulatory drugs (IMiDs) that are commonly used to treat multiple myeloma (MM), the second most frequent hematologic malignancy. Patients respond well to initial treatment with IMiDs, but virtually all patients develop drug resistance over time, and the underlying mechanisms are poorly understood. We identified an as yet undescribed DNA hypermethylation in an active intronic CRBN enhancer. Differential hypermethylation in this region was found to be increased in healthy plasma cells, but was more pronounced in IMiD-refractory MM. Methylation significantly correlated with decreased CRBN expression levels. DNA methyltransferase inhibitor (DNTMi) in vitro experiments induced CRBN enhancer demethylation, and sensitizing effects on lenalidomide treatment were observed in 2 MM cell lines. Thus, we provide first evidence that aberrant CRBN DNA methylation is a novel mechanism of IMiD resistance in MM and may predict IMiD response prior to treatment.

PI3K-dependent multiple myeloma cell survival is mediated by the PIK3CA isoform.

Constitutive phosphatidylinositide 3-kinase (PI3K) signalling has been implicated in multiple myeloma (MM) pathophysiology and is regarded as an actionable target for pharmacological intervention. Isoform-specific PI3K inhibition may offer the most focused treatment approach and could result in greater clinical efficacy and reduced side effects. We therefore performed isoform-specific knockdown of PIK3CA, PIK3CB, PIK3CD, and PIK3CG to analyse their individual contributions to MM cell survival and downstream signalling. In addition, we tested the effectivity of the novel PI3K isoform-specific inhibitors BYL-719 (PIK3CA), TGX-221 (PIK3CB), CAL-101 (PIK3CD), and CAY10505 (PIK3CG). We found the PIK3CA isoform to be of paramount importance for constitutive Akt activity in MM cells, and - in contrast to inhibition of other class I isoforms - only the blockade of PIK3CA was sufficient to induce cell death in a sizeable subgroup of MM samples. Furthermore, pharmacological PIK3CA inhibition in combination treatments of BYL-719 and established anti-myeloma agents resulted in strongly enhanced MM cell death. Our data thus clearly indicate therapeutic potential of PIK3CA inhibitors and support their clinical evaluation in multiple myeloma.

68Ga-Pentixafor PET/CT for Detection of Chemokine Receptor CXCR4 Expression in Myeloproliferative Neoplasms.

C-X-C motif chemokine receptor 4 (CXCR4) is an attractive target for cancer diagnosis and treatment, as it is overexpressed in many solid and hematologic malignancies. This study investigated the feasibility of CXCR4-directed imaging with PET/CT using 68Ga-pentixafor to visualize and quantify disease involvement in myeloproliferative neoplasms (MPNs). Methods: Twelve patients with MPNs (4 with primary myelofibrosis, 6 with essential thrombocythemia, and 2 with polycythemia vera) and 5 controls underwent 68Ga-pentixafor PET/CT. Imaging findings were compared with immunohistochemical stainings, laboratory data, and splenic volume. Results:68Ga-pentixafor PET/CT was visually positive in 12 of 12 patients, and CXCR4 target specificity could be confirmed by immunohistochemical staining. A significantly higher tracer uptake could be detected in the bone marrow of MPN patients (SUVmean, 6.45 ± 2.34 vs. 4.44 ± 1.24). Dynamic changes in CXCR4 expression determined by 68Ga-pentixafor PET/CT corresponded with treatment response. Conclusion:68Ga-pentixafor PET/CT represents a novel diagnostic tool to noninvasively detect and quantify the extent of disease involvement in MPNs.

Bacterial toxins induce sustained mRNA expression of the silencing transcription factor klf2 via inactivation of RhoA and Rhophilin 1.

Yersiniae bearing the Yersinia virulence plasmid pYV impact the transcriptome of J774A.1 macrophage-like cells in two distinct ways: (i) by suppressing, in a Yersinia outer protein P (YopP)-dependent manner, the induction of inflammatory response genes and (ii) by mRNA induction of the silencing transcription factor klf2. Here we show that klf2 induction by Yersinia enterocolitica occurs in several cell lines of macrophage and squamous and upper gastrointestinal epithelial origin as well as in bone marrow-derived dendritic cells. Several strains of Pseudomonas aeruginosa and Staphylococcus aureus are equally effective as Y. enterocolitica in inducing klf2 expression. Screening of mutant strains or incubation with recombinant toxins identified the rho-inactivating toxins YopT from Yersinia spp., ExoS from Pseudomonas aeruginosa, EDIN-B from Staphylococcus aureus, and C3bot from Clostridium botulinum as bacterial inducers of klf2 mRNA. klf2 mRNA induction by these toxins does not require de novo protein synthesis. Serum response factor or actin depolymerization does not seem to be involved in regulating klf2 expression in response to bacterial infection. Instead, short hairpin RNA-mediated inactivation of RhoA and its effector rhophilin 1 is sufficient to induce long-term klf2 expression. Thus, bacteria exploit the RhoA-rhophilin signaling cascade to mediate sustained expression of the immunosuppressive transcription factor klf2.