# Development of multiplex single cell phosphotyrosine profiling tools for B-cell malignancies

> **NIH NIH R21** · UNIVERSITY OF CONNECTICUT SCH OF MED/DNT · 2020 · $178,350

## Abstract

Summary
Aberrant activation of tyrosine kinases causes dysregulated cell signaling resulting in cancer cell proliferation,
invasion, and metastasis. Targeted tyrosine kinase inhibitors are achieving the remarkable success in the
field of hematologic malignancies, exemplifying the significance of studying activated tyrosine kinase
pathways in human diseases. To understand what pathways are active and may thus be targeted
therapeutically, comprehensive analysis of tyrosine phosphorylation of cellular proteins is needed. Currently,
tyrosine phosphoproteomics (pTyr-omics) approaches including mass spectrometry and microarray systems
are predominantly used as research tools rather than for clinical diagnosis due to technical complexities. The
lack of robust and convenient pTyr-omics tools hampers the discovery of biomarkers relevant to tyrosine
kinase pathways and the development of new therapeutic strategies. By contrast, flow cytometry has been
integrated in clinical laboratories as a routine tool for immunophenotyping of blood cells at the single cell level.
In an effort to develop a molecular diagnostic tool based on the global tyrosine phosphorylation state, we
developed SH2 profiling in which SH2 domains, a major pTyr binding module in mammalian cells, are utilized
as probes to detect changes in the global tyrosine phosphorylation state of cancer cells. Recently, we found
that a group of chronic lymphocytic leukemia (CLL) cases selected by SH2 profiling at diagnosis showed high
likelihood of disease progression during ~5 years of clinical follow-up. This result suggests the potential to use
the SH2 profile as a predictive biomarker, motivating us to develop a new assay platform by which the SH2
profiling can be routinely performed in clinical laboratories with single-cell resolution.
Here we propose to develop BCR pathway-focused SH2 profiling assays integrating two new flow cytometry
technologies (SH2-flow). In Aim 1, we will prepare a universal B Cell Receptor (BCR) probe library (BCR-SH2
panel) in which fluorescent dyes are covalently and stoichiometrically coupled to SH2 domains, allowing for
multiplexed binding assays. In Aim 2, we will develop a multi-color flow cytometry assay of human B
lymphocytes using the set of fluorescent SH2 probes. In Aim 3, we will develop an SH2 binding assay based
on CyTOF mass cytometer for highly multiplex SH2 profiling at a single cell resolution. Bringing a pathway-
focused phosphotyrosine profiling tool to routine clinical practice by complementing existing antibody-based
immunophenotyping would have a remarkable impact on clinical research and possibly on patient care. The
new assays could be utilized for discovery of signaling-based predictive biomarkers, indicators for disease
progression, and tools to assess targeted drug responses.

## Key facts

- **NIH application ID:** 10015224
- **Project number:** 5R21CA234824-02
- **Recipient organization:** UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
- **Principal Investigator:** Kazuya Machida
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $178,350
- **Award type:** 5
- **Project period:** 2019-09-10 → 2021-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10015224

## Citation

> US National Institutes of Health, RePORTER application 10015224, Development of multiplex single cell phosphotyrosine profiling tools for B-cell malignancies (5R21CA234824-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10015224. Licensed CC0.

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