Paper Details

ABSTRACT

Myelofibrosis (MF) is an aggressive MPN driven by JAK2, CALR, or MPL mutations and is only cured by allogeneic hematopoietic stem cell transplantation (allo-HCT). Yet, 10-40% of patients relapse post-transplant and determinants of relapse and graft-versus MF (GvMF) remain poorly understood. Here we show that serial spatial molecular analysis of bone marrow (BM) pre- and post-transplant yields new insights into relapse and cure from allo-HCT in MF. We developed a custom in situ genotype-informed single cell spatial transcriptomics platform capable of detecting somatic mutations as well as host versus donor clones directly in archival BM core biopsies. We discovered that MF mutant cells create immunosuppressive local niches driven by TGFb1 that functionally impair cytotoxic CD8 T and NK cells, contributing to advanced disease and post-transplant relapse. We therefore engineered MF antigen-specific T cells with dominant negative TGFb receptors to overcome the immunosuppressive BM microenvironment.

We generated Xenium spatial data across 70 BM cores (~4.7 million cells). In addition to 16 allo-HCT patients with serial sampling pre- and post-allo-HCT, we also profiled 10 untreated and 5 JAK inhibitor treated MF patients. Allo-HCT patients contributed 55 longitudinal cores from day+30 post-transplant (D+30) to D+360. 4 patients relapsed post-transplant, from which we obtained 4-5 longitudinal cores each. 52 cores were from JAK2 mutant patients, 8 were from CALR mutated patients, and 10 from MPL mutated patients.

From this dataset, we identified 28 transcriptionally distinct populations including megakaryocytic, stromal, endothelial, pericyte, adipocyte, osteoclast, and mast cell compartments that are poorly captured by aspirate-based single-cell RNA-seq but central to MF pathogenesis. Probes against sex-specific genes also resolved donor-host chimerism in 11 patients receiving sex-mismatched transplants and were concordant with clinical chimerism testing.

To identify mutant cells at single cell spatial resolution we designed custom probes targeting JAK2 mutant transcripts and key co-occuring somatic mutations (such as in IDH2 and SRSF2). These probes recapitulated paired clinical NGS variant allele fractions and were highly specific for mutant tissue. JAK2-mutant cells exhibited lineage-specific transcriptional remodeling with upregulation of JAK-STAT targets, inflammatory mediators, the profibrotic cytokine TGFβ1, and immune checkpoint/evasion molecules such as CD274, LAG3, and CD47. Neighborhood analysis revealed these mutant cells preferentially occupy a megakaryocyte stromal niche that is markedly expanded in relapsed patients and detectable at lower frequency pre-transplant in individuals destined to relapse. By contrast, erythroid-regenerating niches were enriched in patients achieving durable remission.

Radial immune proximity profiling demonstrated selective depletion of cytotoxic NK and CD8 T cells and enrichment of immunoregulatory programs in immune cells (CD56 bright NK and Treg cells) near mutant JAK2 clones. This was accompanied by distance dependent loss of cytotoxic effector molecules GZMB/GNLY/PRF1 and gain of exhaustion markers. These findings were consistent with a local, TGFβ1-mediated immunosuppressive niche contributing to advanced disease and relapse after transplant.

Consistent with the TGFβ1 immunosuppressive radius surrounding JAK2 mutant cells in MF, enforced TGFβ1 secretion from MPN cell lines attenuated killing of these cells in co-culture with CAR-T cells targeting mutant CALR or TCR T cells targeting minor histocompatibility antigens HA-1 and HA-2. However, this immunosuppressive microenvironment was overcome by introducing a dominant negative (dn)TGFBR2 receptor armor in both CAR-T and TCR-T therapies, resulting in efficient tumor cell killing.

This study establishes a scalable framework for genotype-informed spatial profiling of clinical BM biopsies and identifies a JAK2-dependent, TGFβ1-driven immune architecture in advanced MF. Our data support a model where JAK2 mutant niches create a local microenvironment that functionally suppresses cytotoxic lymphocytes, contributing to post-transplant relapse. By directly visualizing this niche and demonstrating that dnTGFBR2 armored adoptive cell therapies resist TGFβ1 mediated immunosuppression, we provide mechanistic rationale for TGFβ resistant immunotherapies in MF and highlight strategies for the future of GvMF targeted cellular therapies in MPNs.