Genome and clonal hematopoiesis stability contrasts with immune, cfDNA, mitochondrial, and telomere length changes during short duration spaceflight.

Background: The Inspiration4 (I4) mission, the first all-civilian orbital flight mission, investigated the physiological effects of short-duration spaceflight through a multi-omic approach. Despite advances, there remains much to learn about human adaptation to spaceflight's unique challenges, including microgravity, immune system perturbations, and radiation exposure. Methods: To provide a detailed genetics analysis of the mission, we collected dried blood spots pre-, during, and post-flight for DNA extraction. Telomere length was measured by quantitative PCR, while whole genome and cfDNA sequencing provided insight into genomic stability and immune adaptations. A robust bioinformatic pipeline was used for data analysis, including variant calling to assess mutational burden. Result: Telomere elongation occurred during spaceflight and shortened after return to Earth. Cell-free DNA analysis revealed increased immune cell signatures post-flight. No significant clonal hematopoiesis of indeterminate potential (CHIP) or whole-genome instability was observed. The long-term gene expression changes across immune cells suggested cellular adaptations to the space environment persisting months post-flight. Conclusion: Our findings provide valuable insights into the physiological consequences of short-duration spaceflight, with telomere dynamics and immune cell gene expression adapting to spaceflight and persisting after return to Earth. CHIP sequencing data will serve as a reference point for studying the early development of CHIP in astronauts, an understudied phenomenon as previous studies have focused on career astronauts. This study will serve as a reference point for future commercial and non-commercial spaceflight, low Earth orbit (LEO) missions, and deep-space exploration.

Matrix stiffness enhances cancer-macrophage interactions and M2-like macrophage accumulation in the breast tumor microenvironment.

The role of intratumor heterogeneity is becoming increasingly apparent in part due to expansion in single cell technologies. Clinically, tumor heterogeneity poses several obstacles to effective cancer therapy dealing with biomarker variability and treatment responses. Matrix stiffening is known to occur during tumor progression and contribute to pathogenesis in several cancer hallmarks, including tumor angiogenesis and metastasis. However, the effects of matrix stiffening on intratumor heterogeneity have not been thoroughly studied. In this study, we applied single-cell RNA sequencing to investigate the differences in the transcriptional landscapes between stiff and compliant MMTV-PyMT mouse mammary tumors. We found similar compositions of cancer and stromal subpopulations in compliant and stiff tumors but differential intercellular communication and a significantly higher concentration of tumor-promoting, M2-like macrophages in the stiffer tumor microenvironments. Interestingly, we found that cancer cells seeded on stiffer substrates recruited more macrophages. Furthermore, elevated matrix stiffness increased Colony Stimulating Factor 1 (CSF-1) expression in breast cancer cells and reduction of CSF-1 expression on stiffer substrates reduced macrophage recruitment. Thus, our results demonstrate that tissue phenotypes were conserved between stiff and compliant tumors but matrix stiffening altered cell-cell interactions which may be responsible for shifting the phenotypic balance of macrophages residing in the tumor microenvironment towards a pro-tumor progression M2 phenotype. STATEMENT OF SIGNIFICANCE: Cells within tumors are highly heterogeneous, posing challenges with treatment and recurrence. While increased tissue stiffness can promote several hallmarks of cancer, its effects on tumor heterogeneity are unclear. We used single-cell RNA sequencing to investigate the differences in the transcriptional landscapes between stiff and compliant MMTV-PyMT mouse mammary tumors. We found similar compositions of cancer and stromal subpopulations in compliant and stiff tumors but differential intercellular communication and a significantly higher concentration of tumor-promoting, M2-like macrophages in the stiffer tumor microenvironments. Using a biomaterial-based platform, we found that cancer cells seeded on stiffer substrates recruited more macrophages, supporting our in vivo findings. Together, our results demonstrate a key role of matrix stiffness in affecting cell-cell communication and macrophage recruitment.

Paired bone marrow and peripheral blood samples demonstrate lack of widespread dissemination of some CH clones.

Clonal hematopoiesis (CH) represents clonal expansion of mutated hematopoietic stem cells detectable in the peripheral blood or bone marrow through next generation sequencing. The current prevailing model posits that CH mutations detected in the peripheral blood mirror bone marrow mutations with clones widely disseminated across hematopoietic compartments. We sought to test the hypothesis that all clones are disseminated throughout hematopoietic tissues by comparing CH in hip vs peripheral blood specimens collected at the time of hip replacement surgery. Here, we show that patients with osteoarthritis have a high prevalence of CH, which involve genes encoding epigenetic modifiers and DNA damage repair pathway proteins. Importantly, we illustrate that CH, including clones with variant allele frequencies >10%, can be confined to specific bone marrow spaces and may be eliminated through surgical excision. Future work will define whether clones with somatic mutations in particular genes or clonal fractions of certain sizes are either more likely to be localized or are slower to disseminate into the peripheral blood and other bony sites.

Allogeneic TCRαß deficient CAR T-cells targeting CD123 in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a disease with high incidence of relapse that is originated and maintained from leukemia stem cells (LSCs). Hematopoietic stem cells can be distinguished from LSCs by an array of cell surface antigens such as CD123, thus a candidate to eliminate LSCs using a variety of approaches, including CAR T cells. Here, we evaluate the potential of allogeneic gene-edited CAR T cells targeting CD123 to eliminate LSCs (UCART123). UCART123 cells are TCRαßneg T cells generated from healthy donors using TALEN® gene-editing technology, decreasing the likelihood of graft vs host disease. As safety feature, cells express RQR8 to allow elimination with Rituximab. UCART123 effectively eliminates AML cells in vitro and in vivo with significant benefits in overall survival of AML-patient derived xenograft mice. Furthermore, UCART123 preferentially target AML over normal cells with modest toxicity to normal hematopoietic stem/progenitor cells. Together these results suggest that UCART123 represents an off-the shelf therapeutic approach for AML.

Mutant PPM1D- and TP53-Driven Hematopoiesis Populates the Hematopoietic Compartment in Response to Peptide Receptor Radionuclide Therapy.

PURPOSE: Hematologic toxic effects of peptide receptor radionuclide therapy (PRRT) can be permanent. Patients with underlying clonal hematopoiesis (CH) may be more inclined to develop hematologic toxicity after PRRT. However, this association remains understudied. MATERIALS AND METHODS: We evaluated pre- and post-PRRT blood samples of patients with neuroendocrine tumors. After initial screening, 13 cases of interest were selected. Serial blood samples were obtained on 4 of 13 patients. Genomic DNA was analyzed using a 100-gene panel. A variant allele frequency cutoff of 1% was used to call CH. RESULT: Sixty-two percent of patients had CH at baseline. Persistent cytopenias were noted in 64% (7 of 11) of the patients. Serial sample analysis demonstrated that PRRT exposure resulted in clonal expansion of mutant DNA damage response genes (TP53, CHEK2, and PPM1D) and accompanying cytopenias in 75% (3 of 4) of the patients. One patient who had a normal baseline hemogram and developed persistent cytopenias after PRRT exposure showed expansion of mutant PPM1D (variant allele frequency increased to 20% after exposure from < 1% at baseline). In the other two patients, expansion of mutant TP53, CHEK2, and PPM1D clones was also noted along with cytopenia development. CONCLUSION: The shifts in hematopoietic clonal dynamics in our study were accompanied by emergence and persistence of cytopenias. These cytopenias likely represent premalignant state, as PPM1D-, CHEK2-, and TP53-mutant clones by themselves carry a high risk for transformation to therapy-related myeloid neoplasms. Future studies should consider CH screening and longitudinal monitoring as a key risk mitigation strategy for patients with neuroendocrine tumors receiving PRRT.

Patient-Derived Bone Marrow Spheroids Reveal Leukemia-Initiating Cells Supported by Mesenchymal Hypoxic Niches in Pediatric B-ALL.

B-cell acute lymphoblastic leukemia (B-ALL) results from the expansion of malignant lymphoid precursors within the bone marrow (BM), where hematopoietic niches and microenvironmental signals provide leukemia-initiating cells (LICs) the conditions to survive, proliferate, initiate disease, and relapse. Normal and malignant lymphopoiesis are highly dependent on the BM microenvironment, particularly on CXCL12-abundant Reticular (CAR) cells, which provide a niche for maintenance of primitive cells. During B-ALL, leukemic cells hijack BM niches, creating a proinflammatory milieu incompetent to support normal hematopoiesis but favoring leukemic proliferation. Although the lack of a phenotypic stem cell hierarchy is apparent in B-ALL, LICs are a rare and quiescent population potentially responsible for chemoresistance and relapse. Here, we developed novel patient-derived leukemia spheroids (PDLS), an ex vivo avatar model, from mesenchymal stromal cells (MSCs) and primary B-ALL cells, to mimic specialized niche structures and cell-to-cell intercommunication promoting normal and malignant hematopoiesis in pediatric B-ALL. 3D MSC spheroids can recapitulate CAR niche-like hypoxic structures that produce high levels of CXCL10 and CXCL11. We found that PDLS were preferentially enriched with leukemia cells displaying functional properties of LICs, such as quiescence, low reactive oxygen species, drug resistance, high engraftment in immunodeficient mice, and long-term leukemogenesis. Moreover, the combination of PDLS and patient-derived xenografts confirmed a microenvironment-driven hierarchy in their leukemic potential. Importantly, transcriptional profiles of MSC derived from primary patient samples revealed two unique signatures (1), a CXCL12low inflammatory and leukemia expansion (ILE)-like niche, that likely supports leukemic burden, and (2) a CXCL11hi immune-suppressive and leukemia-initiating cell (SLIC)-like niche, where LICs are likely sustained. Interestingly, the CXCL11+ hypoxic zones were recapitulated within the PDLS that are capable of supporting LIC functions. Taken together, we have implemented a novel PDLS system that enriches and supports leukemia cells with stem cell features driven by CXCL11+ MSCs within hypoxic microenvironments capable of recapitulating key features, such as tumor reemergence after exposure to chemotherapy and tumor initiation. This system represents a unique opportunity for designing ex vivo personalized avatars for B-ALL patients to evaluate their own LIC pathobiology and drug sensitivity in the context of the tumor microenvironment.

Targeting the epichaperome as an effective precision medicine approach in a novel PML-SYK fusion acute myeloid leukemia.

The epichaperome is a new cancer target composed of hyperconnected networks of chaperome members that facilitate cell survival. Cancers with an altered chaperone configuration may be susceptible to epichaperome inhibitors. We developed a flow cytometry-based assay for evaluation and monitoring of epichaperome abundance at the single cell level, with the goal of prospectively identifying patients likely to respond to epichaperome inhibitors, to measure target engagement, and dependency during treatment. As proof of principle, we describe a patient with an unclassified myeloproliferative neoplasm harboring a novel PML-SYK fusion, who progressed to acute myeloid leukemia despite chemotherapy and allogeneic stem cell transplant. The leukemia was identified as having high epichaperome abundance. We obtained compassionate access to an investigational epichaperome inhibitor, PU-H71. After 16 doses, the patient achieved durable complete remission. These encouraging results suggest that further investigation of epichaperome inhibitors in patients with abundant baseline epichaperome levels is warranted.

Clonal Hematopoiesis Before, During, and After Human Spaceflight.

Clonal hematopoiesis (CH) occurs when blood cells harboring an advantageous mutation propagate faster than others. These mutations confer a risk for hematological cancers and cardiovascular disease. Here, we analyze CH in blood samples from a pair of twin astronauts over 4 years in bulk and fractionated cell populations using a targeted CH panel, linked-read whole-genome sequencing, and deep RNA sequencing. We show CH with distinct mutational profiles and increasing allelic fraction that includes a high-risk, TET2 clone in one subject and two DNMT3A mutations on distinct alleles in the other twin. These astronauts exhibit CH almost two decades prior to the mean age at which it is typically detected and show larger shifts in clone size than age-matched controls or radiotherapy patients, based on a longitudinal cohort of 157 cancer patients. As such, longitudinal monitoring of CH may serve as an important metric for overall cancer and cardiovascular risk in astronauts.

Fundamental Biological Features of Spaceflight: Advancing the Field to Enable Deep-Space Exploration.

Research on astronaut health and model organisms have revealed six features of spaceflight biology that guide our current understanding of fundamental molecular changes that occur during space travel. The features include oxidative stress, DNA damage, mitochondrial dysregulation, epigenetic changes (including gene regulation), telomere length alterations, and microbiome shifts. Here we review the known hazards of human spaceflight, how spaceflight affects living systems through these six fundamental features, and the associated health risks of space exploration. We also discuss the essential issues related to the health and safety of astronauts involved in future missions, especially planned long-duration and Martian missions.

Plasma Cell Myeloma Presenting With Amyloid-Laden Crystal-Negative Histiocytosis.

OBJECTIVES: Crystal-storing histiocytosis (CSH) is rare in plasma cell dyscrasias, with only 3 cases reported in the setting of amyloid. No cases of crystal-negative histiocytosis coincident with multiple myeloma and amyloidosis have been reported previously. METHODS: A 58-year-old woman presented with pain due to destructive bone lesions and was found to have plasma cell myeloma (PCM) and marrow amyloid deposition associated with crystal-negative histiocytosis. Differential diagnoses included Langerhans cell histiocytosis, Erdheim-Chester disease, and Rosai Dorfman disease. BRAF mutations were negative, and there was no evidence of paraprotein crystals, arguing against typical CSH. RESULTS: The patient was treated with bortezomib, cyclophosphamide, and dexamethasone, and she subsequently underwent autologous stem cell transplant and ixazomib maintenance. She achieved complete remission with improvement of her symptoms and preserved remission after following up at 60 months. CONCLUSIONS: We describe a case of crystal-negative histiocytosis associated with PCM. CSH is a rare disorder associated with paraprotein-producing conditions in which immunoglobulins aggregate as intracellular crystals in the lysosomes of organ-specific phagocytic macrophages. Light chain tropism in PCM can also lead to the development of amyloid deposition in organs and, in rare cases, is associated with light chain aggregation as intracellular crystals in macrophages.

Clonal Hematopoiesis and Premalignant Diseases.

Clonal hematopoiesis (CH) arises when mutations in the hematopoietic system confer a fitness advantage to specific clones, thereby favoring their disproportionate growth. The presence of CH increases with age and environmental exposures such as cytotoxic chemotherapy or radiotherapy. The most frequent mutations occur in epigenetic regulators, such as DNMT3A, TET2, and ASXL1, leading to dysregulation of tumor suppressor function, pathogen response, and inflammation. These dysregulated processes elevate risk of overall mortality, cardiovascular disease, and eventual hematologic malignancy (HM). CH is likely acting as an initiating event leading to HM when followed by cooperating mutations. However, further evidence suggests that CH exerts a bystander influence through its pro-inflammatory properties. Delineating the mechanisms that lead to the onset and expansion of CH as well as its contribution to risk of HM is crucial to defining a management and intervention strategy. In this review, we discuss the potential causes, consequences, technical considerations, and possible management strategies for CH in the context of HMs and pre-HMs.

Cranberry A-type proanthocyanidins selectively target acute myeloid leukemia cells.

Most elderly patients affected with acute myeloid leukemia (AML) will relapse and die of their disease even after achieving complete remission, thus emphasizing the urgent need for new therapeutic approaches with minimum toxicity to normal hematopoietic cells. Cranberry (Vaccinium spp.) extracts have exhibited anticancer and chemopreventive properties that have been mostly attributed to A-type proanthocyanidin (A-PAC) compounds. A-PACs, isolated from a commercially available cranberry extract, were evaluated for their effects on leukemia cell lines, primary AML samples, and normal CD34+ cord blood specimens. Our results indicated potent and specific antileukemia activity in vitro. In addition, the antileukemia activity of A-PACs extended to malignant progenitor and stem cell populations, sparing their normal counterparts. The antileukemia effects of A-PACs were also observed in vivo using patient derived xenografts. Surprisingly, we found that the mechanism of cell death was driven by activation of NF-κB. Overall, our data suggest that A-PACs could be used to improve treatments for AML by targeting leukemia stem cells through a potentially novel pathway.

Clonal Hematopoiesis and risk of Acute Myeloid Leukemia.

Acute Myeloid Leukemia, the most common form of acute leukemia in adults, is an aggressive hematopoietic stem cell malignancy that is associated with significant morbidity and mortality. Though AML generally presents de novo, risk factors include exposure to chemotherapy and/or radiation, as well as both familial and acquired bone marrow failure syndromes. Clonal Hematopoiesis (CH) refers to an expansion of blood or marrow cells resulting from somatic mutations in leukemia-associated genes detected in individuals without cytopenias or hematological malignancies. While CH is considered part of normal ageing, CH is also significantly associated with cardiovascular disease, solid tumors, and hematological malignancies. In this review, we will discuss evidence linking CH with the development of AML, as well as describe challenges in and strategies for monitoring patients with high risk CH mutations.

Randomized trial of 10 days of decitabine ± bortezomib in untreated older patients with AML: CALGB 11002 (Alliance).

Novel treatment strategies are needed for older patients with acute myeloid leukemia (AML). This randomized phase 2 trial compared the efficacy and safety of 20 mg/m2 of IV decitabine on days 1 to 10 alone (arm A) with those of 1.3 mg/m2 of subcutaneous bortezomib (arm B) on days 1, 4, 8, and 11 for up to 4 10-day cycles followed by monthly 5-day cycles. Previously untreated AML patients age ≥60 years (excluding those with FLT3 mutations and favorable-risk cytogenetics) without restrictions in performance status (PS) or organ function were eligible. Median age was 72.4 years (range, 60.5-92.3 years); 31 patients (19%) had baseline PS ≥2, 35 (22%) had an antecedent hematological disorder, 58 had (39%) adverse cytogenetics, and 7 (5%) and 23 (14%) had abnormal cardiac or renal function. There were no statistically significant differences in overall survival (OS) or responses between the 2 treatment arms. The overall response rate (complete remission + complete remission with incomplete blood count recovery) was 39% (n = 64), with median OS of 9.3 months. Nineteen responders (31%) underwent allogeneic stem cell transplantation. The most common adverse event was febrile neutropenia, and there were no unexpected toxicities. Adding bortezomib to decitabine did not improve outcomes, but responses were better than those in previous trials using 5-day decitabine cycles. This trial was registered at www.clinicaltrials.gov as #NCT01420926.

Somatic mutations precede acute myeloid leukemia years before diagnosis.

The pattern of somatic mutations observed at diagnosis of acute myeloid leukemia (AML) has been well-characterized. However, the premalignant mutational landscape of AML and its impact on risk and time to diagnosis is unknown. Here we identified 212 women from the Women's Health Initiative who were healthy at study baseline, but eventually developed AML during follow-up (median time: 9.6 years). Deep sequencing was performed on peripheral blood DNA of these cases and compared to age-matched controls that did not develop AML. We discovered that mutations in IDH1, IDH2, TP53, DNMT3A, TET2 and spliceosome genes significantly increased the odds of developing AML. All subjects with TP53 mutations (n = 21 out of 21 patients) and IDH1 and IDH2 (n = 15 out of 15 patients) mutations eventually developed AML in our study. The presence of detectable mutations years before diagnosis suggests that there is a period of latency that precedes AML during which early detection, monitoring and interventional studies should be considered.