scRNA-seq suggests a new combination therapy recruits stem cell populations favorable for autologous stem cell transplants in cancer patients

Hematopoietic and pluripotent stem cells (HPSCs) mobilization is critical for effective autologous stem cell transplants (ASCT) in multiple myeloma(MM) patients as chemotherapy often depletes these cells, which are mainly responsible for maintaining and repopulating the blood (3). ASCT significantly improves overall survival in patients with MM. While bone marrow and peripheral blood can serve as graft sources, collecting HSPCs from peripheral blood is less invasive and more accessible, making it preferable in many cases (3,4,5).

A recent blog post by 10x Genomics discusses the use of single-cell RNA sequencing (scRNA-seq) to identify a novel combination therapy that recruits stem cell populations favorable for autologous stem cell transplants (ASCT) in patients with multiple myeloma (MM).

The clinical trial evaluated the combination of BioLineRx’s investigational candidate Motixafortide and Filgrastim (G-CSF), a standard drug for HSPC mobilization to assess the safety and efficacy of the combination therapy for ASCT in MM patients (1).

The study’s finding revealed that the motixafortide and filgrastim combination therapy, which was FDA approved (2), was more effective than G-CSF alone in mobilizing HSPCs for ASCT in MM patients. 

Importantly, the researchers employed scRNA-seq analysis of patient samples to obtain the populations of HSPCs most favorable for long-term ASCT success. The analysis helped identify rare cell populations indicating therapeutic responses, biomarkers for patient stratification, and novel mechanisms of action.

How does the mechanism of action of Motixafortide promote HSPC mobilization?

Motixafortide, a peptide inhibitor, promotes HSPC mobilization by binding to the CXCR4 receptor and inhibiting the binding of CXCL2, a molecule that keeps HSPCs in the bone marrow (6). Compared to other CXCR4 inhibitors, motixafortide exhibits high affinity for the receptor and long-lasting effects (1).

Clinical results

The clinical trial included 122 MM patients, and those treated with motixafortide and G-CSF achieved the optimal number of stem cells for ASCT in a shorter time compared to that with placebo and G-CSF group. The durability of grafts, progression-free survival, and overall survival did not show significant differences between the motixafortide + G-CSF and G-CSF alone groups after 100 days post-ASCT. However, the researchers believe that the new mobilization therapy holds potential for improving long-term survival based on scRNA-seq analysis of the clinical trial samples.

The study also identified and characterized different subsets of CD34+ HSPCs. Using techniques like uniform manifold approximation and projection (UMAP) clustering, the researchers distinguished 20 transcriptionally distinct subsets and focused on six subsets with transcriptional profiles resembling hematopoietic stem cells. 

Applications of single cell sequencing

The authors used Chromium Single Cell 5’ Gene Expression to identify the variety and prevalence of CD34+ HSPC subsets particularly primitive HSCs—in clinical samples from 12 people with MM treated with motixafortide + G-CSF, plerixafor + G-CSF, or placebo + G-CSF to understand the mechanism driving improved HSPC mobilization observed with motixafortide + G-CSF in the clinical trial. 

Specifically, the particular stage of each subset during differentiation from a hematopoietic stem cell to a mature lineage cell (e.g., lymphocytes and erythrocytes) is determined. Traditional methods like flow cytometry, which rely on cell surface proteins such as CD34+, are insufficient for identifying transient cell states. For example, a classic diagram of hematopoietic stem cell differentiation often presents various differentiation trajectories, including stem cell differentiation to a lymphoid progenitor, which differentiates into natural killer cells and lymphocytes (see Figure 1). But the arrows pointing from one illustrated cell to another fail to capture the countless, small transcriptional adjustments the cell made to get there. 

Single cell sequencing analyzes transcript levels in individual cells and thus provides necessary resolution to detect these transient states. Trajectory analysis is applied to predict the differentiation process and determine the most transcriptionally primitive cell subset (HSC1) that gave rise to other subsets (HSC2-6 and myeloid progenitor cells) originating from either HSC6 or MLP (myeloid progenitor cells) (7). 

The small adjustments the cells made across these seven cell states added up to big differences in the cell makeup between cohorts and treatments. The proportion of primitive HSC1 cells in samples from healthy donors treated with only motixafortide was more than 2.5% compared to less than 2% in those treated solely with plerixafor or G-CSF. The proportion of HSC1 cells was also higher in people with MM treated with motixafortide + G-CSF than those with plerixafor + G-CSF or placebo + G-CSF (Figure 4). Additionally, differential gene expression analysis also confirmed the HSC1 population was enriched most in those treated with motixafortide due to increased expression of genes previously associated with quiescence, HSC maintenance, and self-renewal. Besides, gene set enrichment analysis corroborated these findings, revealing increased signaling in key pathways involved in hematopoietic regeneration and regenerative potential.

In conclusion, scRNA-seq analysis of MM patient samples in a phase 3 clinical trial revealed the efficacy of the combination therapy between motixafortide and filgrastim in mobilizing HSPCs for ASCT. The study provided valuable insights into the populations of HSPCs most favorable for long-term ASCT success and challenged previous assumptions about CD34+ HSPCs, making a significant step towards improving the outcomes of autologous stem cell transplants.

References:

1. Crees ZD, et al. Motixafortide and G-CSF to mobilize hematopoietic stem cells for autologous transplantation in multiple myeloma: a randomized phase 3 trial. Nat Med 29: 869–879 (2023). DOI: 10.1038/s41591-023-02273-z
2. https://www.prnewswire.com/news-releases/biolinerx-announces-fda-approval-of-aphexda-motixafortide-in-combination-with-filgrastim-g-csf-to-mobilize-hematopoietic-stem-cells-for-collection-and-subsequent-autologous-transplantation-in-patients-with-multiple-myeloma-301923206.html
3. https://www.cancer.gov/about-cancer/treatment/types/stem-cell-transplant#:~:text=in%20clinical%20trials-,Types%20of%20cancer%20treated%20with%20stem%20cell%20transplants,used%20to%20treat%20certain%20cancers
4. Hatzimichael E & Tuthill M. Hematopoietic stem cell transplantation. Stem Cells Cloning 3: 105–117 (2022). DOI: 10.2147/SCCAA.S6815
5. https://ashpublications.org/ashclinicalnews/news/5761/Marrow-or-Peripheral-Blood-for-Allogeneic
6. Broxmeyer HE, et al. Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist. J Exp Med. 201: 1307-18 (2005). DOI: 10.1084/jem.20041385.
7. https://cole-trapnell-lab.github.io/monocle3/docs/trajectories/