Westfall, M., Spector, M. S., Gulrajani, M., Marimpietri, C., Kritharis, A., Fields, S. Z., Allen, S. L., Hawtin, R. (April 2013) Functional characterization of KIT and Fc epsilon R1 receptor mutations in Mast Cell Leukemia using Single Cell Network Profiling (SCNP). Cancer Research, 73 (8 (Sup). p. 3524. ISSN 0008-5472
Abstract
Background: A recent report described an imatinib/dasatinib (Imat/Dasat) resistant MCL patient (pt) with mutations in KIT (V654A) and Fc{varepsilon}R1 (L188F) receptors [Spector et al, Leukemia 2011]. The pt did not respond to cytarabine-based induction therapy combined with Dasat or to post-induction Imat. The functional consequence of the receptor mutations on downstream signaling networks, and sensitivity to alternative therapeutics, was unknown. SCNP is a flow cytometry-based assay that quantitatively and simultaneously measures, in single cells, extracellular surface markers and activation of intracellular signaling proteins in response to modulation. SCNP was applied to interrogate downstream signaling networks and network sensitivity to targeted therapeutics by examining: 1) Basal and modulated signaling using stem cell factor (SCF) or α-IgE 2) Effect on basal and modulated signaling of: a) KIT inhibitors Imat, Dasat, and Nilotinib b) PI3K inhibitor GDC-0941 c) SYK inhibitor fostamatinib R406 Methods: Cryopreserved MCL BMMCs and healthy donor BMMCs were processed alongside fresh healthy donor basophils (FHDB) as controls. BMMCs were modulated with SCF for 5 and 15 min +/-KIT or PI3K inhib. MCL BMMCs and FHDB were modulated with α-IgE for 5min +/- fostamatinib. Signaling in the KIT and Fc{varepsilon}R1 pathways was quantified through measurement of p-AKT/ p-ERK/ p-S6 and p-ERK/ p-PLC{gamma}2 / p-SYK levels, respectively, in the MCL population: CD45+, CD34+, CD33+, and CD117+. Results: As previous reported, the V654A KIT mutation did not result in constitutive activation of the PI3K or MAPK pathways in MCL blasts, but was associated with dysfunctional SCF modulated signaling. Specifically, SCNP identified SCF induced p-AKT levels at 5min, higher (2X) compared to CD34+/CD117+ FHDB, and sustained to 15min with no simultaneous induction of p-ERK or p-S6. Consistent with the clinically observed Imat/Dasat resistance of the MCL case, in vitro AKT induction was unaffected by the presence of KIT inhibitors but sensitive to the PI3K inhib GDC-0941. KIT inhibitors and GDC-0941 blocked SCF induced signaling in the healthy BMMC control. Despite robust p-ERK induction in the FHDB control after α-IgE modulation of the Fc{varepsilon}R1 receptor and inhibition by fostamatinib treatment, no basal or α-IgE modulated Fc{varepsilon}R1 receptor signaling was detected in MCL BMMC cells. Conclusions: SCNP functionally characterizes signaling and drug resistance profiles in MCL BMMCs and can potentially inform on therapeutic selection. Despite robust p-ERK induction in the FHDB control after α-IgE modulation of the Fc{varepsilon}R1 receptor and inhibition by fostamatinib treatment, no basal or α-IgE modulated Fc{varepsilon}R1 receptor signaling was detected in the cryopreserved MCL BMMCs. Further studies will elucidate whether the lack of detected signal could be related to the freeze/thaw process and/or to cell-type specific differences.
| Item Type: | Paper |
|---|---|
| Additional Information: | Meeting Abstract |
| Subjects: | diseases & disorders > cancer > cancer types > leukemia Publication Type > Meeting Abstract bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > mutations bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein receptor |
| CSHL Authors: | |
| Communities: | CSHL labs > Tuveson lab |
| Depositing User: | Matt Covey |
| Date: | April 2013 |
| Date Deposited: | 11 Apr 2014 16:06 |
| Last Modified: | 21 Feb 2018 19:24 |
| URI: | https://repository.cshl.edu/id/eprint/29758 |
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