Manipulating MEF2C: Discovering Novel Drugs to Target ETP-ALL

Ectopic MEF2C expression due to rearrangements have been recurrently found in patients with human early thymocyte progenitor acute lymphoblastic leukemia (ETP-ALL). ETP-ALL is the most immature subtype of T-ALL and is characterized by the expression of myeloid lineage markers and an ETP-like gene expression profile. In normal haematopoiesis, MEF2C expression occurs at immature stages and in myeloid- and B cell lineages but is absent in early T cell progenitors. Here, we investigate MEF2C as an oncogene for ETP-ALL. First, we validated our initial observations in an independent T-ALL cohort that ETP-ALL patients highly express MEF2C along with BCL2, HHEX, LMO2 and LYL1 in contrast to other T-ALL subtypes. In addition, we have demonstrated that enforced MEF2C expression in the T-lineage drives a bi-phenotypic CD3 +CD19 + leukemia in mice by inhibiting a T-cell developmental program in favor a B-cell transcriptional program and that resembles human ETP-ALL. This confirms MEF2C as an oncogenic driver of ETP-ALL. We developed a model system that enabled screening of various drug libraries for compounds that overcome developmental arrest induced by MEF2C-expression, as evidenced by the upregulation of CD3 and TCRγδ. The ETP-ALL-like LOUCY cells express MEF2C as consequence of a del5(q14-qter). Further induction of MEF2C levels (i.e. LOUCY-iMEF2C cells) blocked differentiation of LOUCY cells towards a CD3 +/TCRγδ + phenotype on OP9-DL1 or DLL4-coated plates in contrast to parental LOUCY cells. This model enabled high-throughput screening of drug libraries using a flow cytometry-based assay for drugs that further promote differentiation of LOUCY-MEF2C cells. We identified various epigenetic inhibitors that promoted differentiation of LOUCY-iMEF2C cells. Unlike AML patients, where HDAC4 was shown to play an important role in the activation of MEF2C and the propagation of the leukemia, we found that different HDACs are involved that can block or promote MEF2C function. This suggests that the role of different HDACs in MEF2C regulation is cell-type or disease dependent. In addition, over-expression of MEF2C raised cellular resistance towards prednisolone treatment. Conversely, inhibition of MEF2C sensitized cells to prednisolone cytotoxicity, even in the presence of MEF2C-overexpression. Together, these data show that MEF2C is an oncogenic driver of ETP-ALL, and ectopic expression of MEF2C negatively affects steroid sensitivity. Specific targeting of only those HDACs that promote MEF2C activity may provide new therapeutic options in ETP-ALL by inhibiting MEF2C function and enhancing steroid sensitivity. Disclosures No relevant conflicts of interest to declare.

Response to Steroid and Immunosuppressive Therapies May Predict Post-transplant Recurrence of Focal Segmental Glomerulosclerosis

Recurrence of focal segmental glomerulosclerosis (FSGS) is a major challenge in kidney transplantation. Several clinical factors, including initial steroid sensitivity, have been associated with increased post-transplant FSGS recurrence risk. However, conflicting data have been reported, possibly due to the heterogeneous pathophysiology of FSGS and the lack of genetic testing of FSGS patients. Further, the response to immunosuppressive therapies have not been evaluated. This study aimed to assess the risk factors for post-transplant recurrence in stringently selected patients based on a comprehensive clinicopathological evaluation and genetic testing. Fifty-nine patients aged 1–25 years at FSGS onset who underwent kidney transplantation between 2002 and 2018 were enrolled. Patients with secondary, familial, syndromic, and genetic FSGS and those who did not undergo genetic testing were excluded. Data from 15 kidney transplant recipients were analyzed. Nine (60%) patients experienced post-transplant FSGS recurrence, while six patients did not. The proportion of patients who achieved complete or partial remission with initial steroid and/or additional therapies with immunosuppressive agents and/or plasmapheresis was significantly higher in the FSGS recurrence group than the group without FSGS recurrence (P=0.04). In conclusion, this study suggests that the response to steroid treatment, other immunosuppressive agents, and/or plasmapheresis may predict post-transplant FSGS recurrence.

Salivary Cytokines in Children with Nephrotic Syndrome versus Healthy Children: A Comparative Study

Background: The aims of this study were to compare salivary cytokines and total protein between children with nephrotic syndrome (NS) and healthy children, and to examine whether saliva parameters can differentiate between steroid sensitivity and resistance and between disease remission and relapse. Methods: Twenty-seven children with nephrotic syndrome were classified according to steroid sensitivity and resistance, and disease remission and relapse. Twenty healthy children served as controls. Whole saliva samples were collected from all the participants. Urine and blood tests done on the same day as the saliva collection were recorded. Salivary total protein was quantified using bicinchoninic acid and IFNγ, IL-4, IL-8, IL-6, and IL1β levels using ELISA. Results: The mean ages of the nephrotic syndrome and control groups were 11.3 ± 2.4 and 9 ± 4.2, respectively. Compared to the control group, for the nephrotic syndrome group, total salivary protein was significantly lower, as were the levels of all the cytokines examined except IFNγ. Statistically significant differences were not found in any of the salivary markers examined between the children with nephrotic syndrome who were treatment sensitive (n = 19) and resistant (n = 8). Protein and IL-8 salivary levels were lower in the active (n = 7) than in the remission (n = 20) group. Conclusions: Salivary parameters distinguished children with nephrotic syndrome in relapse from healthy children. This may be due to decreased salivary protein excretion, which reflects decreased plasma levels, consequent to proteinuria. Accordingly, salivary markers may be developed as a diagnostic or screening tool for NS activity.

Photoaffinity labeling identifies an intersubunit steroid-binding site in heteromeric GABA type A (GABAA) receptors

Allopregnanolone (3α5α-P), pregnanolone, and their synthetic derivatives are potent positive allosteric modulators (PAMs) of GABAA receptors (GABAARs) with in vivo anesthetic, anxiolytic, and anti-convulsant effects. Mutational analysis, photoaffinity labeling, and structural studies have provided evidence for intersubunit and intrasubunit steroid-binding sites in the GABAAR transmembrane domain, but revealed only little definition of their binding properties. Here, we identified steroid-binding sites in purified human α1β3 and α1β3γ2 GABAARs by photoaffinity labeling with [3H]21-[4-(3-(trifluoromethyl)-3H-diazirine-3-yl)benzoxy]allopregnanolone ([3H]21-pTFDBzox-AP), a potent GABAAR PAM. Protein microsequencing established 3α5α-P inhibitable photolabeling of amino acids near the cytoplasmic end of the β subunit M4 (β3Pro-415, β3Leu-417, and β3Thr-418) and M3 (β3Arg-309) helices located at the base of a pocket in the β+–α− subunit interface that extends to the level of αGln-242, a steroid sensitivity determinant in the αM1 helix. Competition photolabeling established that this site binds with high affinity a structurally diverse group of 3α-OH steroids that act as anesthetics, anti-epileptics, and anti-depressants. The presence of a 3α-OH was crucial: 3-acetylated, 3-deoxy, and 3-oxo analogs of 3α5α-P, as well as 3β-OH analogs that are GABAAR antagonists, bound with at least 1000-fold lower affinity than 3α5α-P. Similarly, for GABAAR PAMs with the C-20 carbonyl of 3α5α-P or pregnanolone reduced to a hydroxyl, binding affinity is reduced by 1,000-fold, whereas binding is retained after deoxygenation at the C-20 position. These results provide a first insight into the structure-activity relationship at the GABAAR β+–α− subunit interface steroid-binding site and identify several steroid PAMs that act via other sites.

Salivary cytokines in children with nephrotic syndrome versus healthy children: a comparative study

Background: The aims of this study were to compare salivary cytokines and total protein, between children with nephrotic syndrome and healthy children; and to examine whether saliva parameters can differentiate between steroid sensitivity and resistance, and between disease remission and relapse. Methods: Twenty-seven children with nephrotic syndrome were classified according to steroid sensitivity and resistance, and disease remission and relapse. Twenty healthy children served as controls. Whole saliva samples were collected from all the participants. Urine and blood tests done on the same day as the saliva collection were recorded. Salivary total protein was quantified using bicinchoninic acid; and IFNγ, IL-4, IL-8, IL-6 and IL1β levels using ELISA. Results: The mean ages of the nephrotic syndrome and control groups were 11.3±2.4 and 9±4.2, respectively. Compared to the control group, for the nephrotic syndrome group, total salivary protein was significantly lower; as were the levels of all the cytokines examined except IFNγ. Statistically significant differences were not found in any of the salivary markers examined, between the children with nephrotic syndrome who were treatment sensitive (n=19) and resistant (n=8). Protein and IL-8 salivary levels were lower in the active (n=7) than in the remission (n=20) group. Conclusions: Salivary parameters distinguished children with nephrotic syndrome in relapse from healthy children. This may be due to decreased salivary protein excretion, which reflects decreased plasma levels, consequent to proteinuria. Accordingly, salivary markers may be developed as a diagnostic or screening tool for NS activity.