Abstract C049: Implementing human bone marrow organoids to interrogate microenvironmental influences on the efficacy of blood cancer immunotherapies
Authors: Wong Zoë C. , Shen Yuqi , Rampotas Alex , Gannon Isaac , Brierley Charlotte , Benlabiod Camelia , Hayder Nawshad , Murphy Eleanor , Olijnik Aude-Anaïs , Roddie Claire , Pulé Martin , Mead Adam J. , Khan Abdullah O. , Psaila Bethan 
Published: November 2024

Abstract
Cancers perpetuate immunosuppressive niches that fuel progression and hinder the clinical utility of immunotherapies. Pre-clinical validation of immunotherapies often relies on 2D models that lack spatial complexity, or mouse models that do not fully recapitulate the human tumor microenvironment (TME). We recently developed a human induced pluripotent stem cell (iPSC)-derived bone marrow organoid platform, creating a robust system to interrogate cancer-stroma cross talk. Here, we further develop the model to create the first in vitro system for evaluation of blood cancer immunotherapies in a human tissue environment. We focused on targeting myeloproliferative neoplasms (MPNs) – chronic blood cancers affecting ∼400,000 individuals in the US that are currently largely incurable. 1/3 of MPNs are driven by a mutation in calreticulin (mutCALR), and the mutCALR oncoprotein is displayed on the cell surface of disease-initiating stem cells and fibrosis-driving megakaryocytes. MPNs are characterized by pronounced inflammation and fibrosis (“myelofibrosis”). Therapeutic antibodies have entered first-in-human trials, and we recently presented pre-clinical data for the first mutCALR-directed CAR-T cell therapy. However, it is unclear how the TME may limit the efficiency of these agents. To address this, we first tested the utility of the organoid platform to evaluate target killing of leukemic cell lines and cells from patients using the anti-CD33 antibody-drug conjugate (ADC) gemtuzumab. The ADC showed good penetration and potent target cell killing in organoids, with only minimal reduction in efficacy in TGFβ-rich/fibrotic microenvironments. We then developed more sophisticated ‘chimeroids’ comprising the iPSC organoid scaffold engrafted with malignant stem cells plus CAR-Ts. Addition of mutCALR-directed CAR-Ts induced highly selective killing of mutCALR+ stem/progenitor cells with minimal toxicity to non-malignant cells including niche cells (n=5 donors), replicating the highly specific and robust killing seen in 2D cultures. To evaluate the impact of TME perturbations on CAR-T phenotype and function, we generated a scRNAseq dataset of 161,970 cells from ‘healthy’ and ‘myelofibrotic’ organoids engrafted with patient-derived mutCALR+ cells plus CAR-Ts, pre-stimulating organoids with two immunoregulatory cytokines highly abundant in myelofibrosis -TGFβ and Galectin-1. CAR- Ts exposed to mutCALR+ cells showed a 2x expansion of CD8+ effector memory cells, with significant enrichment of IFNγ, TNFα and IL-2 signaling gene-sets. The proportion of immunosuppressive Tregs increased in a high TGFβ TME, but not with Galectin-1. Ongoing analyses are further interrogating crosstalk between CAR-T, target cells, and niche components, and exploring the pro-inflammatory impact of CAR-T killing on the marrow niche – a potential mediator of delayed hematopoietic recovery. These studies demonstrate the utility of human organoids to evaluate blood cancer-targeting immunotherapies within a relevant human TME, with broad relevance for mechanistic and translational studies.
Citation Format: Zoë C. Wong, Yuqi Shen, Alex Rampotas, Isaac Gannon, Charlotte Brierley, Camelia Benlabiod, Nawshad Hayder, Eleanor Murphy, Aude-Anaïs Olijnik, Claire Roddie, Martin Pulé, Adam J. Mead, Abdullah O. Khan, Bethan Psaila. Implementing human bone marrow organoids to interrogate microenvironmental influences on the efficacy of blood cancer immunotherapies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C049.