Erich Piovan (University of Padova, Padova, Italy) for providing the PTEN antibody and Dr

Jul 26, 2022 Phosphatases

Erich Piovan (University of Padova, Padova, Italy) for providing the PTEN antibody and Dr. with other Notch inhibitors. In one model, resistance appeared after 156 days of treatment, it was stable and associated with loss of Notch inhibition, reduced mutational load and acquired mutations potentially affecting the stability of the heterodimerization domain. Conversely, in another model resistance developed after only 43 days of treatment despite persistent down-regulation of Notch signaling and it was FRP accompanied by modulation of lipid metabolism and reduced surface expression of NOTCH1. Our findings shed light on heterogeneous mechanisms adopted by the tumor to evade NOTCH1 blockade and support clinical implementation of antibody-based target therapy for Notch-addicted tumors. Introduction T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological disease that results from clonal expansion of transformed lymphoid progenitors at different developmental stages.1 Cure rates for pediatric ALL are currently approximately 90%, but prognosis for children who experienced relapse remains poor, and it has only marginally improved over the past two decades. Therefore, more efforts are required for patients with chemotherapy-resistant leukemia to identify effective treatment strategies.2,3 The Notch pathway plays a crucial role in T-cell lineage specification and thymic development and its deregulated activation has been linked to T-ALL development and maintenance.1,4 Notably, about 50-60% Galactose 1-phosphate Potassium salt of T-ALL Galactose 1-phosphate Potassium salt samples show activating mutations in the gene5,6 and 15% of T-ALL cases present mutations or deletions in its ubiquitin ligase mutations, including samples derived from relapsed and difficult-to-treat patients.9 OMP52M51 has been in clinical trial in patients with relapsed or refractory lymphoid malignancies (section. Antigens were identified by luminescent visualization using the Western Lightning Plus ECL (Perkin Elmer) or ECL Select (Amersham, GE Healthcare, Chicago, IL, USA) and signal intensity was measured using a Biorad XRS chemiluminescence detection system. In a set of experiments we used subcellular fractionation, which was performed as previously described in Pinazza mutations. Responder PDX disclosed increased T-ALL cell apoptosis, reduction of proliferation and marked inhibition of the Notch transcriptional signature.9 To investigate whether and when resistance to NOTCH1 blockade occurs in a regimen of continuous administration of OMP52M51 mAb, we treated n=3 xenografts bearing activating mutations9 and initially responsive to OMP52M51 (PDTALL8, PDTALL11, PDTALL19) until the appearance of leukemia. For each PDX, leukemia bearing mice (n=5-6 per group) were treated with OMP52M51 or control antibody once a week. Development of leukemia was evaluated by regular blood drawings and flow cytometric analysis of human CD5 and CD7 T-ALL markers and mice were sacrificed when they presented ~20-25% circulating blasts (Figure 1A). Percentages of T-ALL cells in the spleen were evaluated at sacrifice, confirming an almost complete infiltration ( 87%) of this hematopoietic organ by leukemic cells both in control and OMP52M51-resistant mice (alterations It is well known that the PTEN/PI3K/AKT pathway is frequently altered in T-ALL and that PTEN loss is involved in resistance induced by GSI13 and other therapies.14 Therefore, we analyzed the expression of PTEN in the three PDX models. PTEN Galactose 1-phosphate Potassium salt was expressed in all models and resistance was not associated with loss of PTEN, since the protein was detectable at comparable levels in treated and control cells (gene, since mutations in this gene have also been correlated with GSI resistance.7 Sequencing of in PDTALL8, PDTALL11 and PDTALL19 models revealed that neither parental nor resistant cells were harboring a mutated version of FBW7 (for Galactose 1-phosphate Potassium salt WES metrics details), allowing the identification of variants that could be not detected by Sanger sequencing due to a relatively low variant frequency. Cytoscan arrays failed to identify copy number variations associated with resistance to OMP52M51 in PDTALL8 cells (variants found only in OMP52M51 resistant samples. cDNA coordinates, amino acidic changes, VAF, alternative allele depth (AD) and DP are reported. (D) Direct sequencing of exon 26 in a representative control (Ctrl Ab #1) and resistant (OMP52M51 resistant #5) pair. Resistance-related mutations are indicated with the red arrows. (E) Flow cytometric analysis of surface expression of NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL) cells from the spleen of PDTALL8 mice treated with either OMP52M51 or control antibody. Two representative samples per group are shown. To investigate whether these mutations occurred at low level in parental cells, we performed targeted sequencing analysis. All treated xenograft (three replicates/group) presented p.L1585P and p.Q1584H variants in cis (gene, which represent some of the previously described mechanisms of resistance to Notch inhibition by.