Recent epidemiological studies have documented the prevalence of somatic mutations within the cells of the hematopoietic system in healthy individuals. These acquired DNA mutations accumulate with age and, in some instances, can provide a competitive advantage to the mutant cell thus allowing for its clonal expansion. This phenomenon is known as clonal hematopoiesis of indeterminate potential (CHIP). While the mutational landscape of CHIP has only partially been deciphered, some of these clonal expansions can be attributed to somatic mutations in driver genes that are recurrently mutated in blood malignancies. These driver genes include epigenetic regulators (TET2, DNMT3A, ASXL1), spliceosome components (SF3B1, SRSF2), signaling proteins (JAK2), and DNA damage response molecules (TP53, PPM1D).

Studies show that CHIP is associated with an increased risk of all-cause mortality. While there is a marked increase in the frequency of hematological cancer in individuals with CHIP, which is to be expected, the major cause of the increased mortality in these populations appears to be an increase in cardiovascular diseases including coronary heart disease, stroke, and early-onset myocardial infarction. Studies in the applicants’ laboratories have provided evidence for a causal link between CHIP, derived from mutations in TET2, DNMT3A, JAK2, TP53 and PPM1D genes, and cardiovascular, metabolic, and renal pathologies. In some instances, it was shown that the pathological effects of a CHIP driver mutation (TET2, TP53 and PPM1D) could be mitigated with specific anti-inflammatory drugs.

Of particular relevance to the proposed studies, there is an accelerated form of clonal hematopoiesis that is observed in individuals that have undergone myelosuppressive treatment and is referred to as “therapy-related clonal hematopoiesis.” Under these conditions, it has been shown that there are hematopoietic clonal expansions with a very high frequency of mutations in PPM1D andTP53, both of which are classic DNA damage response genes. In individuals undergoing cytotoxic therapy, the hematopoietic system is likely under extreme stress, and it is thought that mutations in genes such as TP53 and PPM1D confer the mutated hematopoietic stem cell with a survival advantage against genotoxic stress induced by chemotherapy. Recent work from the applicants’ laboratories have shown that this form of CHIP can synergize with the genotoxic agent’s direct effect on the cardiovascular system to promote a more robust cardiomyopathic phenotype. While the impact of space travel on CHIP is completely unknown, it is reasonable to speculate that space radiation in combination with other space travel-related stresses will lead to radiation-specific and gene-specific accelerations of clonal hematopoiesis. Further, these forms of CHIP may increase the risk of leukemogenic and cardiovascular pathologies in a radiation- and gene-specific manner.

During the current year, we were able to participate in the Spring and Summer 2023 NASA Space Radiation Laboratory (NSRL) campaigns. In the months leading up to this campaign, we prepared the mice that were used as bone marrow donors. Recipient mice were purchased from Jackson Laboratory. Fourty-eight of these recipient mice received TP53 wild-type bone marrow cells, via the murine adoptive transfer bone marrow transplant (BMT) approach. The re-analysis of this condition was necessary because we discovered that the commercial DNA sequencing service had a mix-up of samples, leading to the “contamination” of wild-type TP53 with mutant TP53 cells used in the BMT. However, these “contaminated” mice will be useful for the final analysis as we were able to determine the ratio of wild-type to mutant cells used for the BMT, and thus this cohort represents a lower gene dosage (that will be informative when compared with the PPMID cohort that is described below). Approximately 2 months after bone marrow transplantation, the mice were transported to Brookhaven National Laboratory (BNL) and exposed to one of four types of radiation: no radiation, 100cGy gamma, 100cGy simGCRsim, or 100cGy SPEsim. One member of the Walsh lab traveled to BNL to complete these irradiation sessions. After the irradiation sessions, the mice were transported back to the University of Virginia, and we are completing serial blood sampling and echocardiography. Currently, this wild-type TP53 cohort is 6 months post radiation exposure. Thus far, in addition to baseline sampling, this cohort of mice has had flow cytometry and whole blood analysis performed at 1 month post-irradiation, and 4 months post-irradiation. Echocardiography was performed before irradiation and approximately every 6 months thereafter. Body weights are measured monthly.

In addition to this cohort, the first TP53 mutant cohort and the PPM1D (mutant and wild-type) cohort have completed the study time course. We also continued to monitor and perform serial sampling for the TET2 (mutant) cohort which is currently 17 months post radiation exposure.

The donor chimerism trends that were noted in the 2022 progress report held true until the study ended. In the male TP53 mutant cohort, the effect of radiation on mutant cell expansion in white blood cells (WBCs) was strongest in the following order: gamma and simGCRsim, followed by SPEsim. In the female mutant cohorts, the effect of radiation was strongest with gamma, and SPEsim and simGCRsim were slightly less. Overall donor chimerism appeared to be greater in the female groups compared to the male groups. Donor chimerism also reached a plateau.

In the PPM1D cohort, the mutant cells did not expand without radiation exposure. The overall percent chimerism was much lower than TP53 and TET2, but the donor cell expansion was still similar to TP53 as a percentage increase. The effect of radiation on mutant cell expansion in WBCs followed similar trends as the TP53 cohort. In both the male and female groups, gamma and simGCRsim showed the greatest expansion of the mutant cells. Similar to what was observed with the TP53 cohort, there appeared to be a sex-specific effect where overall donor chimerism appeared to be greater in the female groups compared to the male groups. Donor chimerism also reached a plateau in the PPM1D cohort.

In the TET2 cohort, the effect of radiation on mutant cell expansion in WBCs still does not appear to be radiation specific. The mutant cell expansion occurs at the same rate regardless of the radiation type. Similar to TP53 and PPM1D there does appear to be a sex-specific effect where there is slightly greater expansion in the female groups. Unlike TP53 and PPM1D donor chimerism does not appear to be reaching a plateau.

Some interesting survival trends also emerged in the TP53, PPM1D, and TET2 cohorts. Survival was substantially lower in the simGCRsim and gamma TP53 mutant male groups. This speed and level of mortality were not seen in either of the other two cohorts. There was also a sex bias where the TP53 mutant males were more affected than the females. There was minimal mortality in the PPM1D cohort, and the mortality did not show a radiation effect, clonal hematopoiesis (CH) mediated effect, or a sex bias. Thus far, TET2 does not appear to have a radiation effect, but there is a CH-mediated effect and a sex bias. Mice in the TET2 knockout groups have higher mortality regardless of radiation type, and mortality in the female KO groups is greater than that in the males.

We also conducted a pilot study to test the hypothesis that gamma radiation would promote the loss of Y chromosome (LOY) clone growth. Male C57Bl6 mice were adoptively transplanted with GFP-positive bone marrow cells with (WT) or without (Y*) the Y chromosome (LOY model). Mice were exposed to 3 doses of 100cGy gamma radiation at 4, 8, and 12 weeks post-adoptive transfer. A subset of mice was not exposed to radiation and served as controls. Blood was collected at 2 and 4 weeks after each radiation dose to assess the percentage of GFP-positive donor white blood (WBCs) cells by flow cytometry. Two weeks after the first radiation dose, the percentage of both WT and Y* donor WBCs was higher in the irradiated groups compared to non-irradiated controls; however, there were no differences between the two irradiated groups. At 4 weeks post-irradiation, the percentage of Y* donor WBCs remained significantly higher in the irradiated group compared to the non-irradiated group. At 2 weeks after the second radiation dose, the percentage of Y* donor WBCs was higher compared to the non-irradiated Y* group; however, there was no difference between the irradiated WT and Y* groups. At 4 weeks after the second radiation dose, there was no difference between any of the groups. At 2 weeks after the third radiation dose, the percentage of Y* donor WBCs was higher compared to the non-irradiated controls, although there was no difference between the WT and Y* irradiated groups. At 4 weeks after the third radiation dose, the percentage of WT donor WBCs was significantly higher than that of Y* in the irradiated groups. While gamma radiation exposure may promote LOY clone expansion to a small extent, this expansion appears to be limited by the number of radiation doses. Moreover, the extent of expansion of LOY cells post-radiation exposure appears to be considerably smaller than that of TP53 and PPM1D mutant clones, as observed in the parent study.

In addition to in vivo experimental models, ongoing cell culture assays are investigating the direct effects of gamma-irradiation mLOY in various cell types. The percentage of mLOY cells will be quantified in primary human fibroblasts and endothelial cells before and after gamma-irradiation treatment, and treated cells will be further expanded in culture and assessed for mLOY over time. These results will determine whether gamma-irradiation has direct effects on the induction and/or expansion of mLOY in model cell types, potentially providing mechanistic evidence for the effects of irradiation on mLOY.

Going forward, we plan to continue flow cytometry and whole blood analysis every 4-6 months and echocardiography every 6-9 months for the TET2 and TP53 cohorts. The mice remaining in the TET2 cohort will be sacrificed and tissues harvested in December 2023 or early January 2024. In planning for future studies, we submitted proposals to irradiate additional cohorts of mice in the Spring and Summer 2024 campaign and the Fall 2024 campaign. Most likely, these new cohorts will examine a model of DNMT3A-mediated clonal hematopoiesis, which is the most prevalent form of clonal hematopoiesis observed in humans.

Recent epidemiological studies have documented the prevalence of somatic mutations within the cells of the hematopoietic system in healthy individuals. These acquired DNA mutations accumulate with age and, in some instances, can provide a competitive advantage to the mutant cell thus allowing for its clonal expansion. This phenomenon is known as clonal hematopoiesis of indeterminate potential (CHIP). While the mutational landscape of CHIP has only partially been deciphered, some of these clonal expansions can be attributed to somatic mutations in driver genes that are recurrently mutated in blood malignancies. These driver genes include epigenetic regulators (TET2, DNMT3A, ASXL1), spliceosome components (SF3B1, SRSF2), signaling proteins (JAK2), and DNA damage response molecules (TP53, PPM1D).

Studies show that CHIP is associated with an increased risk of all-cause mortality. While there is a marked increase in the frequency of hematological cancer in individuals with CHIP, which is to be expected, the major cause of the increased mortality in these populations appears to be an increase in cardiovascular diseases including coronary heart disease, stroke, and early-onset myocardial infarction. Studies in the applicants’ laboratories have provided evidence for a causal link between CHIP, derived from mutations in TET2, DNMT3A, JAK2, TP53 and PPM1D genes, and cardiovascular, metabolic, and renal pathologies. In some instances, it was shown that the pathological effects of a CHIP driver mutation (TET2, TP53 and PPM1D) could be mitigated with specific anti-inflammatory drugs.

Of particular relevance to the proposed studies, there is an accelerated form of clonal hematopoiesis that is observed in individuals that have undergone myelosuppressive treatment and is referred to as “therapy-related clonal hematopoiesis.” Under these conditions, it has been shown that there are hematopoietic clonal expansions with a very high frequency of mutations in PPM1D andTP53, both of which are classic DNA damage response genes. In individuals undergoing cytotoxic therapy, the hematopoietic system is likely under extreme stress, and it is thought that mutations in genes such as TP53 and PPM1D confer the mutated hematopoietic stem cell with a survival advantage against genotoxic stress induced by chemotherapy. Recent work from the applicants’ laboratories have shown that this form of CHIP can synergize with the genotoxic agent’s direct effect on the cardiovascular system to promote a more robust cardiomyopathic phenotype. While the impact of space travel on CHIP is completely unknown, it is reasonable to speculate that space radiation in combination with other space travel-related stresses will lead to radiation-specific and gene-specific accelerations of clonal hematopoiesis. Further, these forms of CHIP may increase the risk of leukemogenic and cardiovascular pathologies in a radiation- and gene-specific manner.

During our second year, we were able to participate in the Spring and Summer 2022 NASA Space Radiation Laboratory (NSRL) campaigns. In the months leading up to this campaign, we prepared the mice that were used as bone marrow donors. Recipient mice were purchased from Jackson Laboratory. Ninety-six of these recipient mice received PPM1D mutant or wild-type bone marrow cells, via the murine adoptive transfer bone marrow transplant (BMT) approach. Another 96 recipient mice received TET2 KO, or wild-type bone marrow cells via the murine adoptive transfer BMT approach. Approximately 2 months after bone marrow transplantation, the mice were transported to Brookhaven National Laboratory (BNL) and exposed to one of four types of radiation: no radiation, 100cGy gamma, 100cGy simGCRsim, or 100cGy SPEsim. One member of the Walsh lab traveled to BNL to complete these irradiation sessions. After the irradiation sessions, the mice were transported back to the University of Virginia, and we are completing serial blood sampling and echocardiography. Currently, the PPM1D cohort is 7 months post-radiation exposure, and the TET2 cohort is 5 months post-radiation exposure. Thus far, in addition to baseline sampling, these cohorts of mice have had flow cytometry and whole blood analysis performed at 1-month post-irradiation, 4 months post-irradiation and then every 3-4 months. Echocardiography was performed before irradiation and approximately every 6 months after that. Body weights are measured monthly. In addition to these 2 cohorts, we continued to monitor and perform serial sampling for the TP53 cohort, which is currently 17 months after radiation exposure.

The longest-running investigation (initiated in 2021) involves the analysis of male and female mice that had undergone adoptive transfer/bone marrow transplantation with the Trp53R270H mutation that is equivalent to the hotspot mutation R273H located at the DNA-binding domain of human TP53 (see Sano et al., JCI Insight, 2021). In the male TP53 cohort, the effect of radiation on mutant cell expansion in white blood cells (WBCs) was strongest in the following order: gamma and simGCRsim, followed by SPEsim. In the female mutant cohorts, the effect of radiation was strongest with gamma, and SPEsim and simGCRsim were slightly less. Overall, donor chimerism appears to be greater in the female groups compared to the male groups.

Based on the ongoing analyses of these TP53 results, we extended the follow-up time from the originally proposed 12 months to approximately 18 months to highlight the age-, radiation-, and sex-dependent phenotypes observed in this cohort.

To date, 21 of 96 mice have been removed from the TP53 cohort. The male mutant gamma and male mutant simGCRsim groups showed decreased survival over the other male mutant groups and all of the female groups. Necropsy of the animals from the TP53 cohort (mostly males) revealed that these mice are expiring from the consequences of hematologic malignancies. A general examination showed that mice exhibit one or more of the following general morphometric phenotypes: enlarged thymus, enlarged abnormally colored liver, enlarged abnormally colored kidneys, enlarged spleen, enlarged lymph nodes, enlarged heart, and obvious anemia. An analysis of blood cell counts over time (up to 12 months post-irradiation—with more time points to follow) revealed one or more of the following phenotypes: severe reductions in white blood cells, monocytes, neutrophils and lymphocytes, or large fluctuations in these parameters (increases followed by decreases, etc.), and large increases in eosinophil content. While some mice displayed enlarged and discolored hearts, serial echocardiographic analyses revealed little or no changes in cardiac contractility. Finally, evaluation by Dr. Eric Pietras (Hematology/Oncology, University of Colorado) provided the following potential diagnoses: bone marrow failure with multi-lineage dysplasia, acute myeloid leukemia or myeloproliferative neoplasia, and possible eosinophilic leukemia (rare). It is well known that men have a higher risk of developing hematologic malignancies or a shorter latency from initiation to diagnosis. The mechanism underlying this sex effect is largely unknown. The TP53 adoptive transfer/irradiation model appears to amplify the sex difference, and it may represent a useful model for understanding the molecular basis for these effects.

In the PPM1D cohort, the effect of radiation on mutant cell expansion in WBCs follows similar trends as the TP53 cohort. In both the male and female groups, gamma and simGCRsim show the greatest expansion of the mutant cells. At 0 through 1-month post-irradiation, this expansion is greatest in the myeloid populations, but then at 1-4 months, the expansion is greatest in lymphoid populations (T and B cells). Similar to what was observed with the TP53 cohort, there appears to be a sex-specific effect where overall donor chimerism appears to be greater in the female groups compared to the male groups. To date, no mortality has occurred in the PPM1D cohort.

In the TET2 cohort, the effect of radiation on mutant cell expansion in WBCs does not appear to be radiation specific. The mutant cell expansion occurs at the same rate regardless of the radiation type. To date, no mortality has occurred in the TET2 cohort.

Going forward, we plan to continue flow cytometry and whole blood analysis every 3-4 months and echocardiography every 6-8 months for the PPM1D, TET2, and TP53 cohorts. The mice remaining in the TP53 cohort will be sacrificed, and tissues harvested in December 2022 or early January 2023. It will be of interest to determine whether the PPM1D and TET2 groups exhibit radiation-induced hematologic disorders and whether there is a strong sex bias, as was seen with TP53.

In planning for future studies, we received approval to irradiate another cohort of mice in the Spring/Summer 2023 campaign. Most likely, this new cohort will examine a model of DNMT3A-mediated clonal hematopoiesis, which is the most prevalent form of clonal hematopoiesis observed in humans.

Going forward, we also plan to repeat aspects of the TP53 cohort.

It may also be of interest to examine the effects of mosaic loss of the Y chromosome (mLOY) in hematopoietic cells. mLOY is the most prevalent post-zygotic mutation in men. The prevalence of mLOY increases with age and, in the UK Biobank, ~45% of men exhibit appreciable mLOY by age 70. mLOY has been associated with increased mortality, cancer, and cognitive diseases. Epidemiological studies suggest that the mortality associated with mLOY can largely explain the 5-year difference in lifespan between men and women. Recently, we documented that men with mLOY exhibit an increased risk of mortality from cardiovascular diseases (S. Sano et al, Science. 2022. 377:292). This study also established, for the first time, a murine model of mLOY, and experiments with this model provided mechanistic evidence for a causal relationship between mLOY and morbidity and mortality.

The pathological consequences of LOY secondary to ionizing radiation are completely unknown. Thus, in addition to the proposed NSRL studies, we have been performing gamma-irradiation pilot studies at our facility (UVA) to gain more insight into how mLOY reacts to the stresses of radiation exposure. These new studies represent the first analysis of LOY cell expansion in the murine adoptive transfer model and the first analysis of the effects of radiation on the expansion of LOY hematopoietic cells. The results of this pilot study indicate that hematopoietic mLOY cell expansion may be highly responsive to radiation, indicating that the murine mLOY model may be of interest for future NSRL studies.

Recent epidemiological studies have documented the prevalence of somatic mutations within the cells of the hematopoietic system in healthy individuals. These acquired DNA mutations accumulate with age and, in some instances, can provide a competitive advantage to the mutant cell thus allowing for its clonal expansion. This phenomenon is known as clonal hematopoiesis of indeterminate potential (CHIP). While the mutational landscape of CHIP has only partially been deciphered, some of these clonal expansions can be attributed to somatic mutations in driver genes that are recurrently mutated in blood malignancies. These driver genes include epigenetic regulators (TET2, DNMT3A, ASXL1), spliceosome components (SF3B1, SRSF2), signaling proteins (JAK2), and DNA damage response molecules (TP53, PPM1D).

Studies show that CHIP is associated with an increased risk of all-cause mortality. While there is a marked increase in the frequency of hematological cancer in individuals with CHIP, which is to be expected, the major cause of the increased mortality in these populations appears to be an increase in cardiovascular diseases including coronary heart disease, stroke, and early-onset myocardial infarction. Studies in the applicants’ laboratories have provided evidence for a causal link between CHIP, derived from mutations in TET2, DNMT3A, JAK2, TP53 and PPM1D genes, and cardiovascular, metabolic, and renal pathologies. In some instances, it was shown that the pathological effects of a CHIP driver mutation (TET2, TP53 and PPM1D) could be mitigated with specific anti-inflammatory drugs.

Of particular relevance to the proposed studies, there is an accelerated form of clonal hematopoiesis that is observed in individuals that have undergone myelosuppressive treatment and is referred to as “therapy-related clonal hematopoiesis.” Under these conditions, it has been shown that there are hematopoietic clonal expansions with a very high frequency of mutations in PPM1D andTP53, both of which are classic DNA damage response genes. In individuals undergoing cytotoxic therapy, the hematopoietic system is likely under extreme stress, and it is thought that mutations in genes such as TP53 and PPM1D confer the mutated hematopoietic stem cell with a survival advantage against genotoxic stress induced by chemotherapy. Recent work from the applicants’ laboratories have shown that this form of CHIP can synergize with the genotoxic agent’s direct effect on the cardiovascular system to promote a more robust cardiomyopathic phenotype. While the impact of space travel on CHIP is completely unknown, it is reasonable to speculate that space radiation in combination with other space travel-related stresses will lead to radiation-specific and gene-specific accelerations of clonal hematopoiesis. Further, these forms of CHIP may increase the risk of leukemogenic and cardiovascular pathologies in a radiation- and gene-specific manner.

In planning for future studies, we submitted proposals for the Spring 2022 and Summer 2022 NSRL campaigns. We were unable to submit a proposal for the Fall 2021 campaign because originally the GCR (galactic cosmic radiation) simulator was going to be unavailable for maintenance. When the maintenance schedule changed, and GCR became available during the Fall 2021 campaign, we did not have enough time to prepare donors and complete the bone marrow transplant before that campaign. At the end of October 2021 the Scientific Advisory Committee for Radiation Research approved our proposal to participate in the Spring 2022 NSRL campaign. Currently, we are preparing the Ppm1d mice that will be used as bone marrow donors for this set of experiments. Recipient mice will be ordered from Jackson Laboratory at the first of the year and the bone marrow transplant will take place approximately 2 months before we are scheduled to irradiate during the Spring 2022 campaign. For this Ppm1d cohort we will follow the same sampling schedule as discussed above for the TP53 cohort. In the next few months we hope to receive approval for the Summer 2022 NSRL campaign. As soon as we receive this approval we will start to prepare the Tet2 mice that will be used as bone marrow donors for this set of experiments. We expect this to be around the beginning of March 2022.

In addition to the NSRL studies, we have been performing irradiation pilot studies at our facility to gain more insight into how various genes, including Tp53, Ppm1d, Tet2, and Dnmt3a, react to the stresses of radiation exposure. The findings of these pilot studies will potentially provide direction for the large cohort to be used in NSRL studies.

Recent epidemiological studies have documented the prevalence of somatic mutations within the cells of the hematopoietic system in healthy individuals. These acquired DNA mutations accumulate with age and, in some instances, can provide a competitive advantage to the mutant cell thus allowing for its clonal expansion, a phenomenon known as clonal hematopoiesis of indeterminate potential (CHIP). While the mutational landscape of CHIP has only partially been deciphered, some of these clonal expansions can be attributed to somatic mutations in driver genes that are recurrently mutated in blood malignancies. These driver genes include epigenetic regulators (TET2, DNMT3A, ASXL1), spliceosome components (SF3B1, SRSF2), signaling proteins (JAK2), and DNA damage response molecules (TP53, PPM1D).

Studies show that CHIP is associated with an increased risk of all-cause mortality. While there is a marked increase in the frequency of hematological cancer in individuals with CHIP, which is to be expected, the major cause of the increased mortality in these populations appears to be an increase in cardiovascular diseases including coronary heart disease, stroke, and early-onset myocardial infarction. Studies in the applicants’ laboratories have provided evidence for a causal link between CHIP, derived from mutations in TET2, DNMT3A, or JAK2 genes, and cardiovascular, metabolic, and renal pathologies. In one instance, it was shown that the pathological effects of a CHIP driver mutation (TET2) could be mitigated with a specific anti-inflammatory drug.

Of particular relevance to the proposed studies, there is an accelerated form of clonal hematopoiesis that is observed in individuals that have undergone myelosuppressive treatment and is referred to as “therapy-related clonal hematopoiesis.” Under these conditions, it has been shown that there are hematopoietic clonal expansions with a very high frequency of mutations in PPM1D andTP53, both of which are classic DNA damage response genes. In individuals undergoing cytotoxic therapy, the hematopoietic system is likely under extreme stress, and it is thought that mutations in genes such as TP53 and PPM1D confer the mutated hematopoietic stem cell with a survival advantage against genotoxic stress induced by chemotherapy. Recent work from the applicants’ laboratories have shown that this form of CHIP can synergize with the genotoxic agent’s direct effect on the cardiovascular system to promote a more robust cardiomyopathic phenotype. While the impact of space travel on CHIP is completely unknown, it is reasonable to speculate that space radiation in combination with other space travel-related stresses will lead to radiation-specific and gene-specific accelerations of clonal hematopoiesis. Further, these forms of CHIP may increase the risk of leukemogenic and cardiovascular pathologies in a radiation- and gene-specific manner.