Characterizing the role of Phlda3 in the development of acute toxicity and malignant transformation of hematopoietic cells induced by total-body irradiation in mice

The tumor suppressor p53 is a transcriptional factor that plays a crucial role in controlling acute toxicity and long-term malignant transformation of hematopoietic cells induced by genotoxic stress such as ionizing radiation. Among all transcriptional targets of p53, one gene that is robustly induced by radiation is the pleckstrin homology domain-only protein Phlda3. However, the role that Phlda3 plays in regulating the response of hematopoietic cells to radiation is unknown. Here, using isogenic cell lines and genetically engineered mouse models, we showed that radiation induces Phlda3 in human leukemia cells and mouse normal hematopoietic cells in a p53-dependent manner. However, deletion of the Phlda3 gene did not ameliorate radiation-induced acute hematologic toxicity. In addition, distinct from mice that lose p53, loss of Phlda3 did not alter the latency and incidence of radiation-induced thymic lymphoma in mice. Remarkably, whole-exome sequencing data showed that lymphomas in irradiated Phlda3+/+ mice harbor a significantly higher number of single nucleotide variants (SNVs) and indels compared to lymphomas in irradiated Phlda3+/− and Phlda3−/− littermates. Together, our results indicate that although deletion of Phlda3 does not accelerate the development of radiation-induced thymic lymphoma, fewer SNVs and indels are necessary to initiate lymphomagenesis after radiation exposure when Phlda3 is silenced.


Materials and methods
Mouse strains and procedures.All animal procedures for this study were approved by the Institutional Animal Care and Use Committee (IACUC) at Duke University (protocol number A215-20-11 was approved on November 9, 2020).All methods were performed in accordance with the relevant guidelines and regulations.The study is reported in accordance with ARRIVE guidelines.Mice with germline deletion of Phlda3 (Phlda3 −/− or called Tih1 −/− in the original publication) 22 were kindly provided by Dr. Benjamin Tycko's laboratory and have been maintained on a C57BL/6J background (CD45.2).In this mouse strain, exon 1 of the Phlda3 gene is replaced by a neomycin cassette 22 .B6.SJL mice (CD45.1)were purchased from The Jackson Laboratory (#002,014).Tie2Cre mice were crossed to Rosa26-LSL-mp53 R172H mice (The Jackson Laboratory Strain #:026,283) to generate mice that express a mutant p53 R172H protein in hematopoietic cells 23,24 .Tie2Cre; p53 FL/− mice were characterized previously 23 .Phlda3 +/+ , Phlda3 +/− and Phlda3 −/− littermates around 4 weeks old were used for radiation-induced thymic lymphoma experiments, whereas in other experiments mice around 8 to 12 weeks old were used.Both male and female mice were used for experiments.Mouse genotyping was performed by Transnetyx.
For the acute radiation syndrome experiments, the health status of irradiated mice was monitored by trained laboratory personnel at least 3 times per week.No supportive care was given to mice after irradiation.The criteria for euthanasia were based on two parameters: decreased activity and squinted/closed eyes on a scale of 1-3 as previously described 25 .A mouse receiving a score of 3 in either category was considered moribund and was euthanized by carbon dioxide asphyxiation.To perform complete blood count (CBC), submandibular cheek bleed was performed to collect whole blood into EDTA tubes (Sarstedt).The samples were then run for CBC by the Duke Veterinary Diagnostic Laboratory.
Radiation treatment.Total-body irradiation was performed 50 cm from the radiation source with a dose rate of 204 cGy/min with 320 kVp X-rays, using 12.5 mA and a filter consisting of 2.5 mm Al and 0.1 mm Cu (X-RAD 320 Biological Irradiator, Precision X-ray).The dose rate was measured with an ion chamber by members of the Radiation Safety Division at Duke University.

Gene expression analysis.
RNA was isolated from thymic tissues using Zymo Direct-zol RNA Miniprep Kit or Zymo Quick-RNA MiniPrep Plus Kit (Zymo Research) according to the manufacturer's protocols.cDNA was synthesized using iScript cDNA Synthesis Kit (Bio-Rad).Gene expression for mouse Phlda3 (Mm00449846_ m1) was analyzed using Taqman probes and TaqMan™ Universal PCR Master Mix (ThermoFischer) with Gapdh (Mm99999915_g1) as an internal control.Human gene expression for Phlda1 (Hs00378285_g1), Phlda2 (Hs00169368_m1), and Phlda3 (Hs00385313_m1) were assessed via TaqMan™ Universal PCR Master Mix with Gapdh (Hs02758991_g1) as an internal control.

Quantification of colony-forming cells.
Whole bone marrow cells were harvested following the procedure described previously 3 .2 × 10 4 whole bone marrow cells were plated in MethoCult GF 3434 (Stem cell technologies).Whole bone marrow cells were irradiated about 1 h after plating and the number of colonies was counted 7 days later.A colony is defined as clusters with a minimal of 30 cells.Colonies were counted without knowledge of the genotype or treatment by two observers (SH and C-LL).
Flow cytometry analysis of hematopoietic stem/progenitor cells in the bone marrow.HSPCs were harvested from the bone marrow of unirradiated and irradiated mice and then stained with antibody cocktails described previously 3,26 .Cells were blocked with a rat anti-mouse CD16/32 antibody (BD Pharmingen) and stained with PE-Cy5 conjugated lineage cocktail containing anti-mouse CD3, CD4, CD8, B220, CD11b, Gr-1 and Ter-119 antibodies (eBioscience), anti-mouse CD27 conjugated to APC (Thermo Fisher) and anti-mouse CD201 conjugated to PE (eBioscience).Data were collected from at least 500,000 single cells by FACSCanto (BD Pharmingen) and analyzed by FlowJo (Tree Star, Inc) without knowledge of the genotype or treatment by a single observer (C-LL).

Detection of thymocyte apoptosis in vitro.
Thymocytes were isolated following the procedure described previously 3 .Irradiation was conducted about 1 h after cell culture in vitro.At 24 h after irradiation, thymocytes were collected, washed using PBS supplemented with Ca 2+ and Mg 2+ , and then stained with a FITCconjugated annexin V (BD Pharmigen) and propidium iodide (5 μg/ml, Sigma-Aldrich).Data were collected from at least 20,000 cells by FACSCanto (BD Pharmingen) and analyzed by Flowjo (Tree Star, Inc) without knowledge of the genotype or treatment by a single observer (C-LL).

Sample preparation for whole exome sequencing.
To prepare samples for WES, thymic lymphoma specimens stored in RNAlater (Invitrogen) and matched snap frozen tails were used for DNA extraction.DNA extraction was performed using the DNeasy Blood and Tissue Kit or the AllPrep DNA/RNA Mini Kit (Qiagen) per the manufacturer's guidelines.Hybrid capture was performed according to the manufacturer's instructions using the Twist Mouse Exome Panel (Twist Bioscience) to enrich for 37.7 megabases targeting the mouse exome.Enriched libraries were sequenced on an Illumina NovaSeq 6000 S1 lane with 150 bp paired-end reads.Samples were demultiplexed and FASTQ files were generated using Bcl2Fastq2 (Illumina).
Whole exome sequencing data analysis.Sequencing data were processed according to the Genome Analysis Toolkit best practices to identify single nucleotide variants (SNVs), small insertions and deletions (indels), and somatic copy number alterations (SCNAs) 28 .Raw sequencing data was aligned to the mm10 mouse genome assembly using BWA-ALN (v0.5.9-r16) 29 .Aligned BAM files were preprocessed using Picard tools (v4.1),Vol:.( 1234567890 www.nature.com/scientificreports/and somatic SNVs and indels were called from the paired lymphoma and normal samples using Mutect2 30 .SNVs were filtered to remove variants (1) located in repetitive regions of the genome (http:// www.repea tmask er.org), (2) with more than 1 alternative read in the matched normal sample, (3) with fewer than 2 alternative reads in the lymphoma sample, and (4) an allele fraction of less than 5%.The Ensembl Variant Effect Predictor (VEP) was used to estimate the impact of SNVs and indels on protein function 31 .SCNAs were identified using CNVkit to integrate on-target and off-target sequencing reads 32 .All normal samples were used as a pooled background reference.SCNAs were filtered to remove copy number variants: (1) present within the matched normal sample and ( 2) with an absolute log 2 copy number ratio less than 0.2.For plotting, cancer-driving genes were defined based on the Catalogue of Somatic Mutations in Cancer (COSMIC) Cancer Gene Census (CGC) 33 .
Statistics.Data are presented as mean ± SEM.Student's t-test (two-tailed) was performed to compare the means of two groups, and one-way ANOVA was performed to compare the means of three or more groups.For survival studies, Kaplan-Meier analysis was performed followed by the log-rank test.To analyze WES data, Twosided Fisher's exact tests were used to compare the proportion of lymphomas from each genotype with SNVs/ indels or SCNAs in each Tier 1 COSMIC gene.Only genes altered in at least 1 lymphoma were included in the analysis, and P-values were corrected for multiple hypothesis testing using the Benjamini-Hochberg procedure.The total number of each type of mutation was compared across lymphomas from mice of each genotype using Kruskal-Wallis tests followed by Dunn's tests for pairwise comparisons.Statistical significance was assumed at P < 0.05.Statistical analyses were performed with Prism 9 (GraphPad Software) or R version 4.1.2through the RStudio environment.

Induction of Phlda3 by ionizing radiation in hematopoietic cells is p53-dependent.
To determine the role of p53 in controlling the induction of PHLDA family genes in human and mouse hematopoietic cells following ionizing radiation, we first examined mRNA expression of PHLDA1, PHLDA2 and PHLDA3 in isogenic human MOLM13 acute myeloid leukemia cells that harbor wild type p53 (p53 +/+ ), no p53 (p53 −/− ) or a p53 R175H mutant protein (p53 R175H/− ) after exposure to 0 or 2.5 Gy X-rays.This radiation dose was selected because it has been shown that radiation doses of 2.5 Gy or less induce strictly p53-dependent cell death 34,35 .
Our results showed that 2.5 Gy significantly induced the expression of PHLDA3 mRNA in MOLM13 p53 +/+ cells 2 and 4 h post-irradiation, whereas the induction was abrogated in p53 −/− and p53 R175H/− counterparts.In contrast, the expression of PHLDA1 and PHLDA2 was not significantly induced by 2.5 Gy in any of the p53 +/+ , p53 −/− or p53 R175H/− MOLM13 cells (Fig. 1A-C and Figure S1).Of note, the expression of PHLDA3 protein was below the detection level, while the protein expression of p53 and p21, a canonical transcriptional target of p53 36 , was induced by 2.5 Gy only in MOLM13 p53 +/+ cells (Figure S2).We also examined mRNA expression of Phlda3 in whole bone marrow cells of Tie2Cre; p53 FL/− mice, in which both alleles of p53 are deleted in hematopoietic cells 23 as well as in Tie2Cre; Rosa26-LSL-mp53 R172H/+ mice, which express a p53 R172H mutant protein (an equivalent of human p53 R175H ) in the hematopoietic system, and their littermate controls (no Cre; p53 +/+ ).Our data showed that 2.5 Gy significantly induced the expression of Phlda3 mRNA in bone marrow cells harvested from p53 +/+ , but not from Tie2Cre; p53 FL/− and Tie2Cre; LSL-mp53 R172H/+ mice (Fig. 1D).Together, our findings indicate that among all three PHLDA family genes, PHLDA3 is the only one that is induced by ionizing radiation via a p53-dependent mechanism in hematopoietic cells of mice and humans at the mRNA level.

Loss of Phlda3 does not protect hematopoietic cells from radiation. To investigate the role of
Phlda3 in vivo, we utilized mice in which the Phlda3 gene is deleted in the germline 22 .All Phlda3 +/+ , Phlda3 +/− and Phlda3 −/− littermates developed normally and showed no notable difference in complete blood counts (Supplementary Table 1).To determine the impact of Phlda3 loss on the response of HSPCs to radiation, we conducted colony-forming cell (CFC) assays using whole bone marrow cells harvested from Phlda3 +/+ , Phlda3 +/− and www.nature.com/scientificreports/Phlda3 −/− mice.Whole bone marrow cells plated in methylcellulose-based media were irradiated with 0 and 2 Gy X-rays in vitro.Although 2 Gy irradiation significantly reduced the number of CFCs derived from bone marrow cells, loss of Phlda3 did not alter the number of CFCs in vitro (Fig. 2A).In addition, we examined the role of Phlda3 in regulating the radiation response of HSPCs in vivo by assessing the number of Lin − CD27 + CD201 + cells 37 .We did not observe a significant difference in the number of Lin − CD27 + CD201 + cells in the bone marrow 24 h after 2.5 Gy TBI between Phlda3 +/+ and Phlda3 −/− mice (Fig. 2B).In addition, we performed competitive repopulation assays using total bone marrow cells harvested from Phlda3 +/+ and Phlda3 −/− littermates 35 days after 2.5 Gy TBI to assess long-term engraftment of irradiated HSPCs 38,39 (Figure S3A).Our findings from competitive repopulation assays using either a 1:1 or a 5:1 ratio of irradiated (CD45.2) to unirradiated (CD45.1)whole bone marrow revealed that the deletion of Phlda3 did not significantly improve long-term hematopoietic reconstitution of irradiated HSPCs (Figure S3B and C).Moreover, we examined the role of Phlda3 in the development of H-ARS by exposing Phlda3 +/+ , Phlda3 +/− and Phlda3 −/− mice to lethal doses of TBI.Our results from the 30-day follow-up did not show significant difference in the overall survival between Phlda3 +/+ , Phlda3 +/− and Phlda3 −/− littermates after 6.5 Gy TBI (Fig. 2C).In addition to bone marrow cells, we also examined radiationinduced apoptosis of thymocytes harvested from Phlda3 +/+ , Phlda3 +/− and Phlda3 −/− mice in vitro.Exposure of thymocytes to 2 and 4 Gy X-rays caused a significant increase in the percentage of Annexin V + cells 24 h after irradiation.However, deletion of Phlda3 did not prevent radiation-induced apoptosis of thymocytes in vitro (Figure S4).Together, these findings demonstrate that although Phlda3 is induced by radiation in hematopoietic cells, deletion of the Phlda3 gene does not protect mice from radiation-induced acute hematopoietic injury.

Loss of Phlda3 does not alter the formation of radiation-induced thymic lymphoma.
To determine the role of Phlda3 in regulating the development of hematologic malignancies following TBI, we exposed Phlda3 +/+ , Phlda3 +/− and Phlda3 −/− littermates to 1.8 Gy TBI every week for 4 consecutive weeks (1.8 Gy × 4).This radiation protocol is robust and highly potent to induce thymic lymphoma that resembles human T-cell lymphoblastic lymphoma/leukemia in wild-type mice 40,41 .Exposure to 6 Gy X-rays (a biological equivalent dose close to 1.8 Gy × 4) significantly increased Phlda3 mRNA expression in Phlda3 +/+ and Phlda3 +/− thymocytes 4 h after irradiation (Figure S5).However, our results showed that the incidence and latency of radiation-induced thymic lymphoma were not statistically different among Phlda3 +/+ , Phlda3 +/− and Phlda3 −/− littermates (Fig. 3A).
As the controls for this experiment, we also examined the impact of deleting p53 or Puma on radiation-induced lymphomagenesis.Consistent with published data 11,13,42 , we observed that the development of radiation-induced thymic lymphoma was substantially accelerated in mice with one copy of p53 (p53 +/− ) compared to their p53 +/+ littermates (Fig. 3B).On the other hand, deletion of Puma significantly protected mice from the formation of radiation-induced thymic lymphoma (Fig. 3C).Together, our findings demonstrate that loss of Phlda3 does not alter the formation of radiation-induced thymic lymphoma in mice.

Mutational landscape in Phlda3 wild-type and Phlda3 deficient radiation-induced lymphomas.
To explore whether loss of Phlda3 affects the mutational spectrum within radiation-induced thymic lymphomas, we performed whole-exome sequencing of radiation-induced lymphomas from Phlda3 −/− , Phlda3 +/− , and Phlda3 +/+ (WT) mice and identified SNVs, indels, and SCNAs.Consistent with our previous work 40 , we observed frequent activating mutations in the Notch1 pathway across genotypes (Fig. 4A, Supplementary Tables S2 and S3).Of the 24 lymphomas analyzed, 20 (83%) had mutations in Notch1 or Ikzf1.However, there was no significant difference in Notch1 pathway mutations across genotypes.Similarly, we did not observe any significant differences in COSMIC Tier 1 genes affected by SNVs/indels or SCNAs between lymphomas in Phlda3 −/− , Phlda3 +/− , and Phlda3 +/+ mice (Supplementary Tables S4 and S5).Of note, we did not observe Phlda3 mutations in any of the lymphomas.We next explored whether loss of Phlda3 affects the number of genomic alterations in radiation-induced lymphomas.We observed significantly more SNVs and indels in Phlda3 +/+ compared with Phlda3 −/− and Phlda3 +/− lymphomas (Fig. 4B).When analyzing only SNVs and indels predicted to have a moderate or high impact on protein function, there continued to be significantly more SNVs and indels in Phlda3 +/+ mice (Fig. 4C).In contrast, there was no significant difference in the total number of SCNAs across genotypes (Fig. 4D).The fact that both Phlda3 −/− and Phlda3 +/− lymphomas showed a distinct phenotype compared to Phlda3 +/+ lymphomas prompted us to examine the expression of Phlda3 mRNA in these tumors.We observed that Phlda3 is not expressed in Phlda3 −/− lymphomas, which confirmed the complete loss of the Phlda3 gene in these tumors.Unexpectedly, we noticed that the expression of Phlda3 mRNA in Phlda3 +/− lymphomas was approximately 20 folds lower compared to which in Phlda3 +/+ lymphomas (Figure S6A).However, SCNA analysis indicated that the substantial decrease in mRNA expression of Phlda3 in Phlda3 +/− lymphomas cannot be explained by copy number alterations of Phlda3 (Figure S6B).Together.our results from WES experiments indicate that fewer SNVs and indels are necessary to initiate lymphomagenesis after radiation exposure when Phlda3 is silenced.

Discussion
Our data reveal that among three PHLDA family genes, Phlda3 is the only one that is induced by radiation in human acute myeloid leukemia cells in a p53-dependent manner.This p53-dependent mechanism of Phlda3 induction is also conserved in mouse bone marrow cells (Fig. 1).These findings indicate that Phlda3 is a specific marker of p53 activation in human and mouse hematopoietic cells.Of note, we attempted to detect the expression of PHLDA3 protein in MOLM13 p53 +/+ AML cells by Western blotting without success (data not shown).These findings suggest the possibility that either the half-life of PHLDA3 protein is short or the level of PHLDA3 protein is below the detection limit.In addition, while our data suggested that PHLDA1 mRNA was not induced by ionizing radiation in MOLM13 p53 +/+ AML cells, this PHLDA family gene has been identified as a transcriptional target of p53 in multiple human solid cancer cell lines 43 .Intriguingly, we observed that the expression of PHLDA1 mRNA at the baseline appears to be even higher in MOLM13 p53 −/− AML cells compared to MOLM13 p53 +/+ AML cells.Thus, it warrants further investigations to elucidate how PHLDA1 expression is regulated in human AML cells.
Although it has been speculated that induction of Phlda3 would promote p53-dependent apoptosis following radiation in normal tissues, our results demonstrate that deletion of Phlda3 neither protects bone marrow-derived HSPCs from acute radiation injury nor prevents the development of H-ARS in mice (Fig. 2).One limitation of our study is the use of mice in which Phlda3 is deleted from the germline because it is possible that hematopoietic cells in Phlda3 −/− mice have adapted the loss of Phlda3.This hypothesis could be tested by conducting experiments using mice in which Phlda3 can be deleted or knocked down in somatic cells acutely before irradiation.Nevertheless, our results indicate that, in contrast to mice with germline deletion of p53 2 or PUMA 9,10 , deletion of Phda3 has very minimal impact, if any, on the response of hematopoietic cells to ionizing radiation in mice.
The tumor suppressor p53 prevents malignant transformation through complex mechanisms including apoptosis, ferroptosis, cell cycle arrest, genomic integrity, metabolism, redox biology, and stemness 44 .One elegant study using mice that harbor mutations in either or both of the transactivation domains of p53 suggested that Phlda3 is a key transcriptional target of p53 that mediates tumor suppression 14 .In addition, it has been reported that Phlda3 is a putative tumor suppressor in pancreatic neuroendocrine tumors 45 .However, using a mouse model of radiation-induced thymic lymphoma, which has been robustly used to study p53-mediated tumor suppression, our results show that deletion of Phlda3 does not alter the incidence and latency of thymic lymphomas induced by 1.8 Gy × 4 TBI (Fig. 3).While loss of p53 results in a substantial increase in the number of genes affected by SCNA in murine radiation-induced thymic lymphomas 12,40 , SCNA is not significantly different among thymic lymphomas in irradiated Phlda3 +/+ , Phlda3 +/− and Phlda3 −/− mice.Moreover, we did not detect any somatic mutations in the Phlda3 gene among all murine thymic lymphomas we examined.Also, examination of human diffuse large B cell lymphoma (DLBCL) dataset from The Cancer Genome Atlas (TCGA) indicate that there are no somatic mutations in PHLDA3 46 .
Our findings from WES show that lymphomas in irradiated Phlda3 +/+ mice harbor a significantly higher number of SNVs and indels compared to lymphomas in irradiated Phlda3 +/− and Phlda3 −/− littermates (Fig. 4).Although mechanisms underlying this phenomenon remain to be further investigated, these data suggest similar processes during thymic lymphomas development between Phlda3 +/− and Phlda3 −/− mice.Indeed, gene expression analysis shows that the expression of Phlda3 mRNA in Phlda3 +/− lymphomas is not significantly higher than in Phlda3 −/− lymphomas.However, WES data reveal that the suppression of Phlda3 mRNA in Phlda3 +/− lymphomas cannot be explained by somatic mutations or copy number changes (Figure S6).These results suggest that the wild type allele of Phlda3 in Phlda3 +/− lymphomas is silenced through epigenetic mechanisms.Of note, human pancreatic neuroendocrine tumors that lose one copy of the Phlda3 allele exhibit frequent promoter hypermethylation of the remaining wild type Phlda3 gene 45 .Thus, it warrants further investigations to examine alterations of genespecific and global methylation among thymic lymphomas in irradiated Phlda3 +/+ , Phlda3 +/− and Phlda3 −/− mice.Collectively, our findings using Phlda3 knockout mice demonstrate that loss of Phlda3 does not alter development of acute hematopoietic injury and thymic lymphoma induced by TBI.However, fewer SNVs and indels are necessary to initiate lymphomagenesis after radiation exposure when Phlda3 is silenced.

Figure 1 .
Figure 1.Ionizing radiation induces Phlda3 mRNA in hematopoietic cells in a p53-dependent manner.(A-C) Isogenic Human MOLM13 acute myeloid leukemia cells that express wild type p53 (WT), no p53 (null) or a p53 R175H mutant protein (R175H) were exposed to 0 or 2.5 Gy X-rays.Cells were harvested 2 h after irradiation to detect the expression of Phlda1, Phlda2 and Phlda3 mRNA.Data are presented as mean ± SEM.N = 3 independent experiments per group.*P < 0.05 by Student's t-test compared to 0 Gy.(D) Whole bone marrow cells harvested from mice expressing wild type p53 (WT), no p53 (null) or a p53 R172H mutant protein (R172H) were exposed to 0 or 2.5 Gy X-rays.Cells were harvested 4 h after irradiation to detect the expression of Phlda3 mRNA.Data are presented as mean ± SEM.N = 3 to 4 mice per genotype.*P < 0.05 by Student's t-test compared to 0 Gy.

Figure 4 .
Figure 4. Whole exome sequencing of radiation-induced lymphomas in Phlda3 wild-type and Phlda3 deficient mice.(A) Plot of genomic alterations identified by whole exome sequencing of radiation-induced thymic lymphomas in Phlda3 −/− , Phlda3 +/− , and Phlda3 +/+ (WT) mice.The top panel shows the tumor suppressors and oncogenes most frequently altered by moderate or high impact SNVs or indels.The bottom panel shows the tumor suppressors and oncogenes with the most frequent SCNAs.Box plots displaying (B) the total number of SNVs/indels, (C) the number of moderate/high impact SNVs/indels, and (D) the total number of SCNAs per lymphoma by mouse genotype.Box shows quartiles and whiskers extend to the minimum and maximum value.P values were calculated using Kruskal-Wallis tests followed by Dunn's tests for pairwise comparisons.