Page 158 - Haematologica May 2020

V. Agnusdei et al.
activating mutations in the NOTCH1 gene5,6 and 15% of T-ALL cases present mutations or deletions in its ubiqui- tin ligase FBW7.7 The role of Notch in solid and hemato- logical malignancies suggests targeting of this pathway for therapeutic purposes in Notch-driven malignancies. Consolidated therapeutic approaches include the use of gamma secretase inhibitors (GSI) that block Notch pro- cessing, alternative molecules that affect Notch signaling and antibodies or decoy peptides to target specific Notch receptors or their ligands, hypothetically overcoming adverse effects due to the pan-Notch signaling inhibition associated with GSI.8
We have recently shown the therapeutic efficacy of the NOTCH1-specific monoclonal antibody OMP52M51 (Brontictuzumab) in pediatric T-ALL xenografts bearing NOTCH1/FBW7 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 (trial ID: NCT01703572) and with relapsed or refractory advanced solid tumors (trial ID: NCT01778439), although it has not been considered for further clinical development. Preliminary experiments in our lab showed that T-ALL PDX acquire resistance to OMP52M51 and this event may likely occur also in patients enrolled in clinical trials with this Notch inhibitor. Moreover, state-of-the-art knowledge about the molecular mechanisms of resistance to Notch blockade stems from various experimental mod- els which utilized almost exclusively GSI to block Notch signaling.10 We were therefore interested in exploiting PDX models to investigate possible novel mechanisms of resistance to antibody-mediated NOTCH1 blockade with the long-term aim to support clinical development of antibodies targeting Notch in cancer.
Methods
Establishment of T-ALL xenografts and development of resistant PDX in vivo
Xenografts (PDTALL) establishment and their genetic charac- terization are reported elsewhere.9 To develop resistance to Notch blockade, PDTALL cells were intravenously (i.v.) injected in NOD/SCID mice (5x106 cells/mouse; 5-6 mice/group) and animals were intraperitoneally (i.p.) treated with the humanized anti-human NOTCH1 mAb OMP52M51 (Oncomed Pharmaceuticals Inc., Redwood, CA, USA) or control antibody (Rituximab, Roche, Basel, Switzerland) until disease progres- sion. Both antibodies were administrated weekly at 20 mg/Kg, starting two days after i.v. injection of T-ALL cells. In the acute treatment experiment, mice were treated with OMP52M51 four days prior to sacrifice. Procedures involving animals conformed current laws and policies (EEC Council Directive 86/609, OJ L 358, 12/12 1987) and were authorized by the Italian Ministry of Health (894/2016-PR). T-ALL growth was monitored by periodic blood drawings and flow cytometric analysis of CD5 and CD7 (the antibodies used were both from Coulter, Fullerton, CA, USA). Leukemic cells were recovered from the spleen and used for the following analyses.
Reverse transcription-PCR (RT-PCR) and quantitative PCR (qPCR)
Total RNA was isolated using TRIzol Reagent (Life Technologies, Paisley, UK) according to the manufacturer’s instructions. cDNA was synthesized from 1-1.5 mg of total RNA
using the High Capacity RNA-to-cDNA kit (Life Technologies). For analysis of the Notch pathway activation, a panel of 21 NOTCH-target genes were evaluated in duplicates by Custom TaqManArray Cards as described before9 using the TaqMan Universal PCR Master Mix (Life Technologies) and ABI Prism 7900 Sequence Detection System. Relative quantification was done using the DDCt method, normalizing to β2-microglobulin mRNA. Primers used for validation of microarray results are reported in the Online Supplementary Material and Methods.
Western blot analysis
Western blot methods used in this study have been previously published.11 Immunoprobing was performed using antibodies reported in the Online Supplementary Material and Methods sec- tion. 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 et al.12
Preparation of cRNA, GeneChip microarray analysis and data normalization
Samples were prepared with GeneChip® WT PLUS Reagent Kit (Affymetrix, Santa Clara, CA, USA). Total RNA and cRNA quality were evaluated with the Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) before microarray hybridization. cDNA was quanti- fied by Implen NanoPhotometer (Implen, München, Germany). Labeled sense-strand cDNA was used for screening of GeneChip® Human Transcriptome Array 2.0 (Affymetrix). Each sample consisted of a single mouse (n=4-5 samples/group). Hybridization and scanning were conducted on the Affymetrix platform. Further experimental details can be found in the Online Supplementary Material and Methods.
Metabolomic analysis
Experimental details can be found in the Online Supplementary Material and Methods.
NGS analysis and Sanger sequencing
Experimental details can be found in the Online Supplementary Material and Methods.
Results
Resistance to OMP52M51 occurs with heterogeneous kinetics and Notch signaling activity in T-ALL PDX
We previously demonstrated that treatment with OMP52M51 delays engraftment of T-ALL cells bearing NOTCH1/FBW7 mutations. Responder PDX disclosed increased T-ALL cell apoptosis, reduction of proliferation and marked inhibition of the Notch transcriptional signa- ture.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 NOTCH1 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
1318
haematologica | 2020; 105(5)