# Novel regulation of Notch-induced HSPC expansion

> **NIH NIH R01** · FRED HUTCHINSON CANCER CENTER · 2020 · $10,234

## Abstract

Project Summary
Our laboratory has developed novel culture methods using engineered Notch ligands to increase the number
of CD34+ precursors, including those able to provide rapid hematopoietic engraftment in patients undergoing
cord blood (CB) transplantation. Clinical trials indicate the need to derive greater hematopoietic stem and
progenitor cell (HSPC) numbers for a more potent, economically feasible therapy. Consequently, this grant
aims to elucidate new strategies to regulate Notch-induced signal strength to maximize HSPC growth, focusing
on exploiting differences in 1) Notch receptor susceptibility to activation among HSPC subsets and 2) Notch
signal strength necessary to promote HSPC expansion versus T cell differentiation. Towards this goal, we
have determined that antibodies raised against the amino-terminus of the Notch extracellular domain are more
effective than Notch ligands in activating receptors on primitive HSPC, leading to increased generation of in
vivo repopulating cells; preliminary studies have suggested this results from antibody-mediated subversion of
inhibitory effects imposed by endogenously expressed ligands in primitive HSPC (cis-inhibition). Thus, in Aim
1, we test our hypothesis that differential agonist potency in promoting Notch activation and HSPC generation
results from ligand-mediated cis-inhibition. We will investigate inhibitory ligand expression and function in CB
HSPC using shRNA knockdown and/or blocking antibody treatment together with transplantation assays. To
elucidate the mechanism whereby antibody subverts cis-inhibition, guiding the future development of improved
agonists, we will determine the contribution of target epitope location and increased agonist affinity. Our
previous studies have also determined requirements for distinct levels of Notch signaling for generation of
HSPC versus promotion of T cell differentiation. Thus, in Aim 2, we test our hypothesis that the use of paralog-
specific Notch antibody agonists will fine-tune the level of Notch activation to concurrently enhance the
generation of marrow and thymic repopulating cells. We will investigate the dynamic use of low densities of
Notch paralog-specific antibody agonists to further increase marrow repopulating cell generation by inducing
Notch activation in cis-inhibited primitive precursors, yet maintain a low level of Notch signal strength to
prevent diversion of these cells to the T-lineage. We further explore whether paralog-specific antibody agonists
can simultaneously generate marrow and thymic repopulating cells by inducing different levels of Notch
activation in different hematopoietic subsets. Overall, these studies will provide insight into the cell-
autonomous mechanisms regulating Notch receptor function, an outcome of conceptual and practical interest
to those focused on manipulating cell-fate decisions in Notch-dependent stem cell types, most relevant here,
the expansion of HSPC and prothymocyte precursors for the devel...

## Key facts

- **NIH application ID:** 10595335
- **Project number:** 6R01DK110563-05
- **Recipient organization:** FRED HUTCHINSON CANCER CENTER
- **Principal Investigator:** IRWIN D BERNSTEIN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $10,234
- **Award type:** 6
- **Project period:** 2017-07-18 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10595335

## Citation

> US National Institutes of Health, RePORTER application 10595335, Novel regulation of Notch-induced HSPC expansion (6R01DK110563-05). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10595335. Licensed CC0.

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