Conditional Knock-in Mouse Model Research Articles

Anthracyclines disaggregate and restore mutant p63 function: a potential therapeutic approach for AEC syndrome

Ankyloblepharon-Ectodermal Defects-Cleft Lip/Palate (AEC) syndrome is a rare genetic disorder caused by mutations in the TP63 gene, which encodes a transcription factor essential for epidermal gene expression. A key feature of AEC syndrome is chronic skin erosion, for which no effective treatment currently exists. Our previous studies demonstrated that mutations associated with AEC syndrome lead to p63 protein misfolding and aggregation, exerting a dominant-negative effect. By performing a high-throughput screening of epigenetic and FDA-approved compounds in a co-transfection model of wild-type and mutant p63, we found that two compounds, Doxorubicin and Epirubicin, alleviate protein aggregation and restore p63 transactivation function. Moreover, treatment with these compounds reduced protein aggregation and restored the expression of keratinocyte-specific p63 target genes in primary keratinocytes derived from a conditional ΔNp63αL514F knock-in AEC mouse model, which mimics the ectodermal defects and skin erosions characteristic of AEC syndrome. A chemical analog of Doxorubicin, diMe-Doxorubicin, which exhibits lower tissue and organ toxicity, was also found to be effective in promoting the disaggregation of mutant p63 and rescuing its transcriptional activity. Our findings identify compounds that can partially resolve mutant p63 aggregation, increase its monomeric isoform, and reactivate its transcriptional function. These results suggest potential therapeutic efficacy for treating skin erosions in AEC syndrome.

Anti-proteolytic regulation of KRAS by USP9X/NDRG3 in KRAS-driven cancer development

Cancers with activating mutations of KRAS show a high prevalence but remain intractable, requiring innovative strategies to overcome the poor targetability of KRAS. Here, we report that KRAS expression is post-translationally up-regulated through deubiquitination when the scaffolding function of NDRG3 (N-Myc downstream-regulated gene 3) promotes specific interaction between KRAS and a deubiquitinating enzyme, USP9X. In KRAS-mutant cancer cells KRAS protein expression, downstream signaling, and cell growth are highly dependent on NDRG3. In conditional KrasG12D knock-in mouse models of pancreatic ductal adenocarcinoma, Ndrg3 depletion abolishes Kras protein expression and suppresses intraepithelial neoplasia formation in pancreas. Mechanistically, KRAS protein binds to the C-terminal serine/threonine-rich region of NDRG3, subsequently going through deubiquitination by USP9X recruited to the complex. This interaction can be disrupted in a dominant-negative manner by a C-terminal NDRG3 fragment that binds KRAS but is defective in USP9X binding, highly suppressing KRAS protein expression and KRAS-driven cell growth. In summary, KRAS-driven cancer development critically depends on the deubiquitination of KRAS protein mediated by USP9X/NDRG3, and KRAS-addicted cancers could be effectively targeted by inhibiting the KRAS-NDRG3 interaction.

Cyclin-dependent kinase inhibitor p18 regulates lineage transitions of excitatory neurons, astrocytes, and interneurons in the mouse cortex.

Neural stem cells (NSCs) can give rise to both neurons and glia, but the regulatory mechanisms governing their differentiation transitions remain incompletely understood. Here, we address the role of cyclin-dependent kinase inhibitors (CDKIs) in the later stages of dorsal cortical development. We find that the CDKIs p18 and p27 are upregulated at the onset of astrocyte generation. Acute manipulation of p18 and p27 levels shows that CDKIs modulate lineage switching between upper-layer neurons and astrocytes at the transitional stage. We generate a conditional knock-in mouse model to induce p18 in NSCs. The transcriptomic deconvolution of microdissected tissue reveals that increased levels of p18 promote glial cell development and activate Delta-Notch signaling. Furthermore, we show that p18 upregulates the homeobox transcription factor Dlx2 to subsequently induce the differentiation of olfactory bulb interneurons while reducing the numbers of upper-layer neurons and astrocytes at the perinatal stage. Clonal analysis using transposon-based reporters reveals that the transition from the astrocyte to the interneuron lineage is potentiated by p18 at the single-cell level. In sum, our study reports a function of p18 in determining the developmental boundaries among different cellular lineages arising sequentially from NSCs in the dorsal cortex.

A new Col1a1 conditional knock-in mouse model to study osteogenesis imperfecta.

Osteogenesis imperfecta (OI) constitutes a family of bone fragility disorders characterized by both genetic and clinical heterogeneity. Several different mouse models reproduce the classic features of OI, and the most-commonly studied carry either a spontaneous or genetically induced pathogenic variant in the Col1a1 or Col1a2 gene. When OI is caused by primary alterations of type I collagen, it represents a systemic connective tissue disease that, in addition to the skeleton, also affects several extra-skeletal tissues and organs such as skin, teeth, lung, heart, and others, where the altered type I collagen is also expressed. Currently, existing mouse models harbor a disease-causing genetic variant in all tissues and do not allow assessing primary versus secondary consequences of the mutation on a specific organ/system. Here, we describe the generation of the first conditional knock-in allele for Col1a1 that can express a severe OI-causing glycine substitution (p.Gly1146Arg) in the triple helical region of α1(I) but only after Cre-driven recombination in the tissue of choice. We called this new dominant allele Col1a1G1146R-Floxed/+ and introduced it into the murine model. We describe its validation by crossing mice carrying this allele with EIIA-Cre expressing mice and showing that offspring with the recombined allele reproduce the classic features of a severe form of OI. The new mouse model will be useful to study the tissue-specific impact of this severe mutation on organs such as the lung, the heart and others.

High mobility group A1 (HMGA1) promotes the tumorigenesis of colorectal cancer by increasing lipid synthesis

Metabolic reprogramming is a hallmark of cancer, enabling tumor cells to meet the high energy and biosynthetic demands required for their proliferation. High mobility group A1 (HMGA1) is a structural transcription factor and frequently overexpressed in human colorectal cancer (CRC). Here, we show that HMGA1 promotes CRC progression by driving lipid synthesis in a AOM/DSS-induced CRC mouse model. Using conditional knockout (Hmga1△IEC) and knock-in (Hmga1IEC-OE/+) mouse models, we demonstrate that HMGA1 enhances CRC cell proliferation and accelerates tumor development by upregulating fatty acid synthase (FASN). Mechanistically, HMGA1 increases the transcriptional activity of sterol regulatory element-binding protein 1 (SREBP1) on the FASN promoter, leading to increased lipid accumulation in intestinal epithelial cells. Moreover, a high-fat diet exacerbates CRC progression in Hmga1△IEC mice, while pharmacological inhibition of FASN by orlistat reduces tumor growth in Hmga1IEC-OE/+ mice. Our findings suggest that targeting lipid metabolism could offer a promising therapeutic strategy for CRC.

ENL mutation and AML: a new model that reveals oncogenic condensate's function in leukemogenesis.

Precise regulation of gene expression is essential for proper development and the maintenance of homeostasis in organisms. Studies have shown that some transcriptional regulatory proteins influence gene expression through the formation of dynamic, locally concentrated assemblies known as condensates, while dysregulation of transcriptional condensates was associated with several cancers, such as Ewing sarcoma and AML [Wang Y etal. (2023) Nat Chem Biol 19, 1223-1234; Chandra B etal. (2022) Cancer Discov 12, 1152-1169]. Mutations in the histone acetylation "reader" eleven-nineteen-leukemia (ENL) have been shown to form discrete condensates at endogenous genomic targets, but it remains unclear how ENL mutations drive tumorigenesis and whether it is correlated with their condensate formation property. Liu etal. now show, using a conditional knock-in mouse model, that ENL YEATS domain mutation is a bona fide oncogenic driver for AML. This mutant ENL forms condensates in hematopoietic stem/progenitor cells at the genomic loci of key leukemogenic genes, including Meis1 and Hoxa cluster genes, and disrupting condensate formation via mutagenesis impairs its chromatin and oncogenic function. Furthermore, they show that small-molecule inhibition of the acetyl-binding activity displaces ENL mutant condensates from oncogenic target loci, and this inhibitor significantly impairs the onset and progression of AML driven by mutant ENL in vivo.

Condensate-Promoting ENL Mutation Drives Tumorigenesis In Vivo Through Dynamic Regulation of Histone Modifications and Gene Expression.

Gain-of-function mutations in the histone acetylation "reader" eleven-nineteen-leukemia (ENL), found in acute myeloid leukemia (AML) and Wilms tumor, are known to drive condensate formation and gene activation in cellular systems. However, their role in tumorigenesis remains unclear. Using a conditional knock-in mouse model, we show that mutant ENL perturbs normal hematopoiesis, induces aberrant expansion of myeloid progenitors, and triggers rapid onset of aggressive AML. Mutant ENL alters developmental and inflammatory gene programs in part by remodeling histone modifications. Mutant ENL forms condensates in hematopoietic stem/progenitor cells at key leukemogenic genes, and disrupting condensate formation via mutagenesis impairs its chromatin and oncogenic function. Moreover, treatment with an acetyl-binding inhibitor of the mutant ENL displaces these condensates from target loci, inhibits mutant ENL-induced chromatin changes, and delays AML initiation and progression in vivo. Our study elucidates the function of ENL mutations in chromatin regulation and tumorigenesis and demonstrates the potential of targeting pathogenic condensates in cancer treatment. Significance: A direct link between ENL mutations, condensate formation, and tumorigenesis is lacking. This study elucidates the function and mechanism of ENL mutations in leukemogenesis, establishing these mutations as bona fide oncogenic drivers. Our results also support the role of condensate dysregulation in cancer and reveal strategies to target pathogenic condensates.

The IFITM5 mutation in osteogenesis imperfecta type V is associated with an ERK/SOX9-dependent osteoprogenitor differentiation defect.

Osteogenesis imperfecta (OI) type V is the second most common form of OI, distinguished by hyperplastic callus formation and calcification of the interosseous membranes, in addition to the bone fragility. It is caused by a recurrent, dominant pathogenic variant (c.-14C>T) in interferon-induced transmembrane protein 5 (IFITM5). Here, we generated a conditional Rosa26-knockin mouse model to study the mechanistic consequences of the recurrent mutation. Expression of the mutant Ifitm5 in osteo-chondroprogenitor or chondrogenic cells resulted in low bone mass and growth retardation. Mutant limbs showed impaired endochondral ossification, cartilage overgrowth, and abnormal growth plate architecture. The cartilage phenotype correlates with the pathology reported in patients with OI type V. Surprisingly, expression of mutant Ifitm5 in mature osteoblasts caused no obvious skeletal abnormalities. In contrast, earlier expression in osteo-chondroprogenitors was associated with an increase in the skeletal progenitor cell population within the periosteum. Lineage tracing showed that chondrogenic cells expressing the mutant Ifitm5 had decreased differentiation into osteoblastic cells in diaphyseal bone. Moreover, mutant IFITM5 disrupted early skeletal homeostasis in part by activating ERK signaling and downstream SOX9 protein, and inhibition of these pathways partially rescued the phenotype in mutant animals. These data identify the contribution of a signaling defect altering osteo-chondroprogenitor differentiation as a driver in the pathogenesis of OI type V.

TIF1β activates leukemic transcriptional program in HSCs and promotes BCR::ABL1-induced myeloid leukemia.

TIF1β/KAP1/TRIM28, a chromatin modulator, both represses and activates the transcription of genes in normal and malignant cells. Analyses of datasets on leukemia patients revealed that the expression level of TIF1β was increased in patients with chronic myeloid leukemia at the blast crisis and acute myeloid leukemia. We generated a BCR::ABL1 conditional knock-in (KI) mouse model, which developed aggressive myeloid leukemia, and demonstrated that the deletion of the Tif1β gene inhibited the progression of myeloid leukemia and showed longer survival than that in BCR::ABL1 KI mice, suggesting that Tif1β drove the progression of BCR::ABL1-induced leukemia. In addition, the deletion of Tif1β sensitized BCR::ABL1 KI leukemic cells to dasatinib. The deletion of Tif1β decreased the expression levels of TIF1β-target genes and chromatin accessibility peaks enriched with the Fosl1-binding motif in BCR::ABL1 KI stem cells. TIF1β directly bound to the promoters of proliferation genes, such as FOSL1, in human BCR::ABL1 cells, in which TIF1β and FOSL1 bound to adjacent regions of chromatin. Since the expression of Fosl1 was critical for the enhanced growth of BCR::ABL1 KI cells, Tif1β and Fosl1 interacted to activate the leukemic transcriptional program in and cellular function of BCR::ABL1 KI stem cells and drove the progression of myeloid leukemia.

Abstract 6234: Single-cell analysis of state-transition from health to leukemia and the leukemic cell heterogeneities in inv(16) acute myeloid leukemia (AML)

Abstract AML is an aggressive hematological malignancy with heterogeneous genetic abnormalities. Using a conditional knock-in mouse model (Cbfb56M/+/Mx1-Cre) to mimic the somatic acquisition of fusion gene CBFB-MYH11 (CM), created in the recurrent chromosome translocation inv(16)(p13q22) of AML, we previously reported that the blood transcriptome undergoes state-transition from health to leukemia. In this study, we analyze the transcriptomic alterations and leukemic cell heterogeneities in a leukemia state-space using single-cell RNA sequencing (scRNA-seq). CM expression was induced in Cbfb56M/+/Mx1-Cre mice by poly (I:C) injection, and scRNA-seq analysis was performed on peripheral blood mononuclear cells (PBMC) and bone marrow (BM) cells from leukemic mice and age-matched wild-type (WT) controls. More than 105,000 cells were subjected to the following dimensionality reduction and cell type annotation. By performing principal component analysis (PCA), we observed a Y-shape cell distribution with emergence of a divergent branch occupied by leukemic cells which can be mapped to a leukemic state. The principal components 1 and 2 (PC1 and PC2) clearly distinguish the leukemic cells from normal cell types such as myeloid cells and lymphocytes. By accessing the eigenvalue of genes for PC1 and PC2, we quantified the crucial genes and pathways related to leukemia progression at a single-cell level. We characterized cKit+ leukemic cell clusters by transcriptome-based correlation and gene expression pattern and found that they resembled stem cells and megakaryocytic-erythroid progenitors (MEP) with dysregulated pathways, including upregulated "inflammatory response" and downregulated pathways related to Myc targets, oxidative phosphorylation, mitochondrial protein translation, and ribosomal biogenesis/assembly. Examining the 31 clusters (C0-C30) seen in cKit+ BM cells, we identified 9 leukemic clusters exhibiting distinct features, including high heme-metabolism (C0, C1, and C2), high proliferation (C1, C8), S phase (C4, C10), low oxidative phosphorylation (C2, C5, C17), high Cd9 (C10), high Egfl7 (C7, C8). In conclusion, our study unveils state-transition from health to leukemia at single-cell resolution, and uncovers diverse biological heterogeneities associated with the inv(16) AML state. Further refinement of transcriptome state-transition and single-cell analysis will provide an analytical framework for modeling the dynamic changes during disease evolution at the single-cell level. Citation Format: Yu-Hsuan Fu, Lianjun Zhang, Ying-Chieh Chen, David E. Frankhouser, Lisa Uechi, Denis O'Meally, Sergio Branciamore, Guido Marcucci, Russell Rockne, Ya-Huei Kuo. Single-cell analysis of state-transition from health to leukemia and the leukemic cell heterogeneities in inv(16) acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6234.

Role of the S1P Signaling Pathway in the Pathogenesis of Angioimmunoblastic T-Cell Lymphoma

Peripheral T cell lymphomas (PTCLs) are a group of aggressive lymphoid malignancies originating from mature T cells. Angioimmunoblastic T-cell lymphomas (AITL) represent 20% to 30% of all PTCL diagnoses and are associated with autoimmune features, poor response to chemotherapy, and dismal prognosis. Molecular profiling of AITL has identified T-follicular helper (TFH) cells as the cell of origin of AITL, while mutational analysis has identified frequent alterations in epigenetic regulators ( TET2, DNMT3A, and IDH2) and elements of the TCR pathway as drivers of AITL transformation. Our group identified the highly recurrent RHOA G17V mutation as a defining hallmark of AITL and other T-cell lymphomas of TFH-cell origin (Palomero et al., 2014). Using a novel conditional knockin mouse model, we demonstrated that expression of Rhoa G17V in CD4+ T-cells induces TFH cell specification and promotes AITL lymphomagenesis in the context of loss of Tet2 (Cortes et al., 2018). Recently, we have identified that Rhoa G17V regulates the expression of the sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) one of the five members of the receptor family of S1P, which plays an essential role in immune response and lymphocyte trafficking. During thymic T-cell development, expression of Rhoa G17V in CD4+ cells inhibited S1PR1 downregulation after the double positive stage leading to altered positive selection, reduced CD4+ single positive cell numbers and premature thymic egress of autorreactive T-cells which results in the development of systemic inflammation and autoimmunity. In our Tet2 −/− RHOA G17V AITL tumor model, constitutive activation of S1P receptors supported activation of the STAT3 and NFkB pathway and enhanced migration of malignant cells to peripheral organs. Mechanistically, expression of Rhoa G17V in mature CD4+ T-cells impaired re-phosphorylation of the Ezrin-Radoxin-Moesin (ERM) complex independently of ROCK signaling and led to the activation and increased migration of CD4+ T-cells. Importantly, blockade of S1PR1 signaling by fingolimod induced anti-tumor activity in Tet2 −/− RHOA G17V lymphoma-bearing mice in vivo with significant reductions in tumor burden associated with increased apoptosis and decreased STAT3 phosphorylation. Furthermore, analysis of a panel of primary tumors from AITL patients using a multiplexed staining panel and PhenoImager platform revealed significantly higher S1PR1 expression in AITL tumor cells than normal TFH cells from benign reactive lymph nodes. Our findings highlight the role of S1P signaling pathway in the pathogenesis of RHOA G17V-driven AITLs and the potential therapeutic benefit of targeting this pathway.

Exploring Aging Dependent Adaptations of Splicing Factor Mutations in Clonal Hematopoiesis

Introduction: It has been shown that leukemia-associated mutations can be detected in the blood of healthy individuals and their clonal expansion in hematopoietic stem and progenitor cell (HSPC) populations is referred to as Clonal Hematopoiesis (CH). In one study, a population analysis of a cohort of 17,182 patients performed by Bick et al. [PMID7944936] selected without prior screening for hematological malignancies, indicated that advancing age is directly associated with an increase in CH. When the mutant clone accounts for 4% of the nucleated blood cells, it is then referred to as Clonal Hematopoiesis of Indeterminant Potential (CHIP). CHIP has been classified by the World Health Organization as a premalignant state and risk criteria for neoplastic development. CHIP is highly associated with adverse outcomes including auto-immune or auto-inflammatory conditions, cardiovascular disease, cytopenia, cancers in general, and other hematological neoplasms. The associated hematopoietic neoplasms include myelodysplastic syndromes (MDS), myeloproliferative disorders (MPD), and acute myeloid leukemia (AML). Population studies reveal splicing factor mutations to be among the most common mutations in CHIP and are highly correlated with age and show a high hazard ratio for risk of developing secondary neoplasms including AML. RNA splicing factors U2AF1, SRSF2, and SF3B1 make up core components of the spliceosome and are critical to prevent aberrant splicing. Mutations in these factors have widespread effects implicating a multitude of cellular processes. There are several established mutant splicing factor mouse models that can be utilized; however, many employ non-specific or leaky Cre drivers. Methods: To avoid non-specific and leaky Cre activation during aging, we used a Cre/lox-mediated U2af1(Q157R) conditional knock-in mouse model that does not contain an internal Cre driver. This mouse has a floxed wild-type U2af1 cDNA insert that expresses wild-type U2AF1 under normal circumstances. However, upon the exogenous addition of recombinant Cre the U2af1 cDNA is excised and the U2af1(Q157R) splicing factor mutation in exon 6 is expressed instead. This mouse model also possesses lox-stop-lox-YFP, leading to expression of YFP along with mutant U2AF1(Q157R) when Cre is introduced. We then isolated bone marrow from 6 week (young) and 6 months (aged) floxed Q157R/YFP mice and treated lineage-depleted bone marrow with recombinant Cre protein for two hours, followed by transplantation into mildly conditioned 6 week (young) and 18 month (old) wild-type mice. The percentage of YFP in the peripheral blood is quantified every 3 weeks. Full analyses, including RNA-seq, of the bone marrow progenitor populations after 3 months is in progress. Results and Discussion: Using the previously mentioned bone marrow transplant model our work shows that when a U2AF1 mutation is introduced into young and old recipient mice, there is significant preferential outgrowth when the donor mice are aged (Figure 1). This is indicated by an increase in the ratio of YFP, which represents the U2AF1(Q157R) mutant, to CD45.1 events, which represent an internal engraftment control. This would suggest that the expansion of U2AF1(Q157R) splicing mutant cells seen in clinical data may be the result of intrinsic factors from the aging cell population. Comprehensive analysis of the effects of alternative splicing in this model is ongoing, however, these data provide support for further investigation of the intrinsic effects of splicing factor mutations in an aged context.

Application of State Transition Theory and Treatment Modeling for Predicting Response to Chemotherapy in a Mouse Model of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a cancer of the myeloid line of blood cells, and the cancer originates in the bone marrow (BM) and circulates the body through the peripheral blood (PB), which can rapidly lead to BM failure and death. AML initiation and progression are composed of multiple biological properties, and we have previously shown that the state transition theory, state-space, and a model of the stochastic differential equation (SDE) could predict AML disease evolution using the mRNA of a conditional Cbfb-MYH11 (CM) knock-in mouse model. The model of the SDE represents AML evolution from health to disease as a state transition of the transcriptome state described as a particle undergoing Brownian motion in a double-well quasi-potential, where critical points define states of healthy state, c1, transition to AML, c2, and overt AML, c3. Along with the disease evolution of AML, prediction, and understanding of AML disease responses from chemotherapy aiming to provide optimized therapeutic strategies are required. Analyzing disease responses to chemotherapy from the state transition theory perspective will give us an understanding of post-chemotherapy disease dynamics and useful predictions which have the possibility to optimize treatment processes. To test the applicability of the AML state transition model, a time-series experiment using the conditional CM knock-in mouse model and state-transition modeling were performed. The mouse model was designed to recapitulate the human inv(16) AML, the leukemogenic fusion gene Cbfb-MYH11 (CM) in conditional CM knock-in mice (Cbfb56M/+/Mx1-Cre) cause the development of AML with a median survival of approximately 4 months after induction of CM. To induce CM expression, 6-8 weeks old CM knock-in mice were injected intraperitoneally with polyinosinic-polycytidylic acid [poly (I:C)] (InvivoGen, tlrl-picw-250) at 14 mg/kg/dose every other day for a total of 7 doses. CM leukemic mice were treated with a combination of cytarabine (50mg/kg/day; 5 days) and daunorubicin (1.5mg/kg/day; 3 days) after detection of overt leukemia, which is monitored by circulating leukemia blast (cKit+ > 20%) to model the 7+3 standard of care treatment for newly diagnosed AML. We used three independent experiments of mice as training and test cohorts (Group 1, Group 2, and Group 3). We collected 42 PBMC and 1 BM from CM-induced mice from Group 1 (4 AML and 1 control), 68 PBMC and 2 BM from Group 2 (4 AML), and 64 PBMC from Group 3 (3 AML and 2 control). The samples were collected weekly before, during, and following chemotherapy and subjected to RNA sequencing. The effects of chemotherapy were observed in the AML state-space with chronological time points as the transcriptome particle moving from leukemia state, c3, towards a state of healthy state, c1, crossing the unstable state, c2. The relapse was defined as the time of the first observation after the particle crosses back over unstable state, c2, towards the leukemic state, c3. The 10 AML mice out of 11 AML mice achieved a partial response with a mean time to relapse of 5 weeks. All 11 AML mice showed the anti-correlation between AML state in the state space was anti-correlated with the blast percentages (ckit%). We modified the state transition treatment model by introducing a parameter, γ, and the half-life of cytarabine (31.1 hours) and daunorubicin (21.9 hours). The gamma parameter is a force that works against the double-well quasi potential (γ >1) and represents the anti leukemogenic effects of cytarabine and daunorubicin. Gamma was determined by fitting the model to 6 AML mice data from the training cohort. The value was determined to be γ=2.7, providing a novel quantification of the treatment effect. We performed a mean arrival time analysis using simulations of the state transition treatment model, which accurately predicted the time to relapse with a prediction error of 6.6 days for the 3 AML mice from the test cohort. We could apply the state transition theory and propose the state transition treatment model to simulate the treatment process and the response from chemotherapy. Our model accurately predicted the time to respond to chemotherapy, from leukemia to health, and relapse, from health to leukemia, in the CM mouse model after chemotherapy. The state transition treatment model has implications for improving therapeutic strategies by targeting transcriptome state transition critical points in human AML.

SF3B1 K700E Alters the Splicing of Metabolism and Stress Response Genes, Attenuating the Effect of DNMT3A Mutations in MDS

Myelodysplastic syndromes (MDS) are clonal stem cell disorders associated with low blood counts and a high risk of leukemic transformation. Genomic sequencing studies have demonstrated that MDS develops due to the acquisition of somatic mutations in hematopoietic stem cells (HSCs). Splicing factor 3b subunit 1 ( SF3B1) mutations, one of the most frequent mutations in MDS, are associated with lower-risk disease characterized by ineffective erythropoiesis. Our group and others have shown that mutations in SF3B1 cause aberrant mRNA splicing in human and murine HSCs, an expansion of long-term HSCs (LT-HSCs) in primary mice, but a competitive disadvantage in transplantation assays. DNMT3A, de novo methyltransferase 3A, is an epigenetic regulator mutated in ~10-15% of MDS. DNMT3A mutations are associated with unfavorable outcomes in MDS including a higher risk of leukemia transformation and shorter overall survival. Murine models of Dnmt3a mutations revealed that Dnmt3a-mutant HSCs have increased self-renewal over lineage-specific differentiation. SF3B1 and DNMT3A mutations co-occur more frequently than expected by chance and arise early in MDS pathogenesis, making them attractive therapeutic targets. MDS patients with SF3B1 and DNMT3A mutations have shorter progression-free survival than those with solely SF3B1 mutations. However, the mechanisms by which these mutations cooperate to cause HSC dysfunction and their possible combined role in leukemogenesis are not well understood. To investigate how Dnmt3a and Sf3b1 mutations disrupt HSC function in vivo we crossed two conditional knock-in mouse models of common point mutations in SF3B1 (K700E) and DNMT3A (R787H, murine equivalent to human DNMT3A R882H). We followed monthly blood counts and evaluated stem and progenitor cell composition in the bone marrow at 6 months. We found that Sf3b1/Dnmt3a double mutant mice display a similar phenotype to Sf3b1K700E mice, with a progressive anemia and an expansion in LT-HSCs. However, when assayed for HSC self-renewal potential, whole bone marrow and LT-HSCs isolated from Sf3b1/Dnmt3a mutant mice display increased serial colony formation capacities similar to Dnmt3a mutant hematopoietic stem and progenitor cells (HSPCs, 5 to 6 passages), but have a competitive disadvantage in secondary and tertiary transplant assays that is less severe than Sf3b1K700E HSPCs but distinct from the competitive advantage of Dnmt3a mutant HSPCs. Transcriptionally, double mutant HSCs clustered closer to Sf3b1 HSCs with shared expression of 74% of upregulated genes and 64% of downregulated genes and have decreased expression of stress response and metabolism genes. These results indicate Sf3b1 mutations may mitigate the preleukemic phenotype due to Dnmt3a mutations in MDS. To identify factors regulated by SF3B1 mutations that may mitigate the effect of DNMT3A loss on leukemic progression, we generated isogenic DNMT3A KO and SF3B1 K700E single and double mutant K562 cell lines using CRISPR/Cas9. Transcriptome analysis demonstrated that SF3B1K700E/DNMT3AKO double mutant clones clustered with and shared similar upregulated (69%) and downregulated (62%) genes as SF3B1 single mutant clones. Similar to our findings in murine HSPCs, pathway analysis of SF3B1 and SF3B1/DNMT3A mutant clones displayed enrichment of stress response and metabolic pathways. Alternative mRNA splicing analysis identified 13 events (3 alternative 3' splice site, 9 intron retention, and 1 skipped exon) in 8 genes that were upregulated in DNMT3A KO clones and downregulated in SF3B1 and SF3B1/DNMT3A mutant clones. We confirmed decreased protein expression of these mis-spliced genes in SF3B1 single and double mutant clones, but not DNMT3A KO clones. These genes include components of the integrated stress response pathway, which are also mis-spliced in SF3B1 and SF3B1/DNMT3A mutant MDS patient samples. Taken together our data suggest that aberrant splicing by mutant SF3B1 prevents metabolic reprogramming and decreases leukemic progression caused by DNMT3A mutation in MDS.

AML1-ETO and CCND2 Overexpression Cooperate to Drive AML Initiation and Progression

Objectives: The translocation between chromosome 8 and 21, resulting in the AML1-ETO fusion gene, is one of the most common cytogenetic abnormalities observed in acute myeloid leukemia (AML). However, AML1-ETO alone is insufficient to cause leukemia, additional secondary events are required for leukemogenesis. CCND2 gene, a key member of the CyclinD2-CDK4/6 complex, plays a crucial role as a cell cycle regulator. In hematopoietic malignancies, CCND2 mutations (particularly with a conserved mutated hotspot on Thr280) have been found in AML, particularly in core binding factor leukemias. The mutated CCND2 protein exhibits greater stability and resistance to proteasomal degradation. Previous research had shown that CCND2 was a transcriptional target of AML1-ETO, indicating that AML1-ETO can promote CCND2 expression. However, it remains unclear whether mutated CCND2 can cooperate with the AML1-ETO to drive leukemia initiation and progression. Methods: In our previous study, a conditional AML1-ETO knock-in mouse model was developed, in which the AML1-ETO fusion gene was tagged with m-Cherry. To investigate the role of CCND2 in this model, the coding sequences of both CCND2 wt (wild-type) and CCND2 mut (mutant with Thr280Ala substitution) were cloned into the retroviral vector MSCV-IRES-EGFP. Subsequently, the fetal liver cells (embryonic day=12.5) from AML1-ETO mice were infected with the corresponding retroviruses. Results: Four months post AML1-ETO induction, the percentage of m-Cherry + GFP + cells of the mice in CCND2 wt and CCND2 mut groups began to increase. The results revealed a substantial upregulation of CCND2 mRNA and protein expression in these groups compared to that of the Vec group. Additionally, the CCND2 mut group exhibited even higher CCND2 protein expression due to impaired protein degradation. Moreover, the shortened survival time and pathological findings in mice from the CCND2 wt and CCND2 mut groups confirmed that they were succumbed to leukemia infiltration. To assess the stemness of leukemia stem cells (LSCs), extreme limiting dilution analysis (ELDA) was conducted, and it showed a significant increase in LSC frequency in the CCND2 wt and CCND2 mut groups compared to that of the Vec group. Notably, transplanting AML1-ETO cells alone didn't confer any competitive advantage in sublethally irradiated mice, while the overexpression of CCND2 provided them with the ability to survive. Transcriptome analysis indicated that compared to the Vec group, CCND2 wt and CCND2 mut groups exhibited notable enrichment in OXPHOS, E2F, and G2M gene sets. Interestingly, the mTORC1 signaling pathway was particularly enriched in the CCND2 mut group compared to the other two groups. To verify this speculation, phosphorylated S6 and total S6 protein levels were assessed through Western blotting. The results showed that compared to Vec group, the p-S6/total S6 ratio show a gradual increase in the CCND2 wt and CCND2 mut groups, indicating activation of the mTOR pathway by CCND2 protein. Everolimus, a selective inhibitor of mTOR, was administered to CCND2 wt and CCND2 mut mice at a dose of 5mg/kg via oral gavage (three times a week) for three consecutive weeks. The Everolimus-treated mice exhibited reduced leukemic burden and fewer number of LSCs compared to that of the vehicle-treated group. Additionally, Everolimus restored the non-leukemic cells and extended their survival time in CCND2 wt and CCND2 mut group mice. Conclusion: In our study, a leukemia mouse model with co-expression of AML1-ETO and CCND2 mutation was successfully established, which demonstrated that the introduction of the CCND2 gene into the existing AML1-ETO model can trigger the development of leukemia. It was also confirmed that CCND2 overexpression results in the upregulation of the mTOR pathway, which drove the progression of leukemia. Everolimus effectively improved the outcome of mice with CCND2 wt or CCND2 mut, suggesting its potential as a therapeutic agent for this subtype of leukemia.

Sohlh2 promotes pulmonary fibrosis via repression of p62/Keap1/Nrf2 mediated anti-oxidative signaling pathway

Disturbance in the redox balance of alveolar epithelial cells (AECs) was considered as a causal factor for pulmonary fibrosis. The regulatory mechanisms of redox hemostasis in the development of pulmonary fibrosis remain largely unknown. Using a type II AEC-specific Sohlh2 conditional knock-in (CKI) mouse model, we found that Sohlh2, a basic HLH transcription factor, accelerated age-related pulmonary fibrosis. High-fat diet (HFD) resulted in a tremendous increase in lung inflammation and fibrotic changes in the lung tissues of Sohlh2 CKI mice. Sohlh2 overexpression led to a significant rise of intracellular ROS and apoptosis in the lung, mouse primary AECIIs, and human A549 cells, which was attenuated by ROS inhibitor (NAC). Sohlh2 enhanced oxidative stress via repressing p62/Keap1/Nrf2 mediated anti-oxidative signaling pathway. p62, a direct target of Sohlh2, mediated Sohlh2 effects on ROS generation and apoptosis in A549 cells. Hence, our findings elucidate a pivotal mechanism underlying oxidative stress-induced pulmonary fibrosis, providing a framework for aging-related disorder interventions.

Abstract 5755: TP63 fusions drive enhancer rewiring, lymphomagenesis, and dependence on EZH2

Abstract Recurrent chromosomal rearrangements are a hallmark of hematologic malignancies and play critical roles in pathogenesis. The TP53 analog TP63 is rearranged in 5-10% of diverse subtypes of both aggressive T- and B-cell lymphomas. Patients with TP63-rearranged lymphomas have dismal outcomes, with 5-year overall survival rates between 0-17%, depending on cohorts. The function and mechanisms of TP63 rearrangements and TP63 fusion proteins in tumorigenesis are poorly understood. As a result, attempts to treat these patients to date have been largely empiric. Thus, there is an urgent need to understand how TP63 fusions contribute to tumorigenesis and to translate the findings into novel therapeutic options for these patients. Here, we demonstrated that TP63 fusions are essential for the propagation of T-cell lymphomas (TCLs). Knockdown of TP63 fusions with specific shRNAs in TCL cell lines harboring TP63 fusions suppressed both cell growth in vitro and tumor growth in vivo. Retroviral expression of TBL1XR1-TP63, the most common TP63 fusion, conferred cytokine independence in Ba/F3 cells, consistent with its role as an oncogene. To investigate the role of TP63 fusions in T- and B-cell lymphomagenesis, we engineered a CAG-Loxp-Stop-Loxp-TBL1XR1-TP63 conditional knock-in mouse model and crossed with hCD2-Cre mice. This results in expression beginning during early lymphoid development. As observed in patients, transgenic mice developed multiple subtypes of both T- and B-cell lymphoma. To define the effects and mechanisms of TP63 fusions within T cells, we performed CRISPR scanning, transcriptomic, epigenomic, and proteomic analyses. Our data showed that domains within both the N-terminal TBL1XR1 and C-terminal TP63 portions contribute to the function of this fusion. We found that the N-terminal component of TP63 fusions interacts with components of the NCOR/SMRT complex. At the same time, the C-terminal portion of TP63 (which recapitulates the deltaN-p63 isoform expressed in some carcinomas) interacts with the enhancer modifier KMT2D and its complex members. TBL1XR1-TP63 binds to a novel distal enhancer to drive MYC expression, and thus upregulates the expression of the histone H3K27 methylase EZH2. Finally, we assessed whether EZH2 is a vulnerability of TP63-rearranged lymphomas. We found that knockdown of EZH2 in TP63-rearranged lines significantly impaired cell growth, as did treatment with the EZH2 and 1 dual inhibitor valemetostat. Valemetostat, which is now being tested in patients with lymphoma, counteracted the oncogenic effects of TP63 fusions in multiple preclinical models in vivo. Together, our results identify the TP63 fusion as a highly unique oncogenic driver in lymphomagenesis capable of recruiting multiple epigenetic modifier complexes and inducing a targetable dependence on EZH2. Citation Format: Gongwei Wu, Noriaki Yoshida, Jihe Liu, Xiaoyang Zhang, Yuan Xiong, Tayla Heavican-Foral, Huiyun Liu, Geoffrey Nelson, Lu Yang, Renee Chen, Katherine Donovan, Marcus Jones, Mikhail Roshal, Yanming Zhang, Ran Xu, Ajit Nirmal, Salvia Jain, Catharine Leahy, Kristen Jones, Kristen Stevenson, Natasha Galasso, Nivetha Ganesan, Tiffany Chang, Wen-Chao Wu, Abner Louissaint, Lydie Debaize, Hojong Yoon, Paola Dal Cin, Wing Chan Chan, Shannan Ho Sui Ho Sui, Samuel Ng, Andrew Feldman, Steven M. Horwitz, Mathew Meyerson, Karen Adelman, Eric Fischer, Chun-Wei Chen, David Weinstock, Myles Brown. TP63 fusions drive enhancer rewiring, lymphomagenesis, and dependence on EZH2. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5755.

Abstract 1372: NUDT16L1 promotes colon cancer progression via inhibition of ferroptosis

Abstract Ferroptosis is an iron-dependent oxidative cell death with accumulation of lipid peroxidation and abnormal morphology of mitochondria. Although previous studies have shown that ferroptosis inducers have therapeutic potentials in several treatment-naive and drug-resistant cancer types, lacking systematic analysis of the ferroptosis sensitivity in different cancer types makes it difficult to know which cancer type is suitable for clinical usage and critical regulator determining the ferroptosis sensitivity during cancer progression is still not clear. In this study, colon cancer was identified as one of the ferroptosis-insensitive cancer types by dry and wet bench analyses. Importantly, our large-scale analysis also showed that NUDT16L1 was a novel ferroptosis repressor and overexpressed in colon cancer clinical specimens. Similar findings are also observed in the chemical-induced and genetic mouse models of colon cancer. Furthermore, NUDT16L1 loss-of-function and gain-of-function cell models and its conditional knock-in and knock-out mouse models are used to prove its crucial role in the development of colon cancer. In addition, loss of NUDT16L1 induced several key ferroptosis characteristics and impairment of mitochondrial functions while its restoration attenuated those phenomena in colon cancer cells. Mechanistically, our RNA-Seq and RIP-Seq analyses shown that NUDT16L1 directly interacted and positively regulated several crucial negative regulators of ferroptosis. More interestingly, loss of NUDT16L1 not only restores the sensitivity of ferroptosis inducer but also impairs the function of mitochondria. Finally, a specific NUDT16L1 inhibitor was shown to not only repress colon cancer growth in vitro and in vivo but also induce the ferroptosis in colon cancer. In conclusion, this is the first study to demonstrate that overexpression of NUDT16L1 promotes colon cancer progression by inhibition of ferroptosis and maintenance proper function of mitochondria. Furthermore, specific NUDT16L1 inhibitor might be a promising therapeutic strategy for colon cancer patient in the future. Citation Format: Yi-Syuan Lin, Ya-Chuan Tsai, Chia-Jung Li, Tzu-Tang Wei, Ya-Na Wu, Shang-Rung Wu, Shin-Chin Lin, Shaw-Jenq Tsai, Shih-Chieh Lin. NUDT16L1 promotes colon cancer progression via inhibition of ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1372.

TP63 Fusions Drive Enhancer Rewiring, Lymphomagenesis, and Dependence on EZH2

Recurrent chromosomal rearrangements are a hallmark of hematologic malignancies and play critical roles in pathogenesis. The TP53 analog TP63 is rearranged in 5-10% of diverse subtypes of both aggressive T- and B-cell lymphomas, which is highly unique among chromosomal translocations. Specifically, TP63 rearrangements were found in 5-10% of ALK-negative anaplastic large cell lymphoma (ALK- ALCL), peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) and primary cutaneous ALCL, 7% of mycosis fungoides with large cell transformation (MF-LCT), and 1% of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Patients with TP63-rearranged lymphomas have dismal outcomes, with 5-year overall survival rates between 0-17%, depending on cohorts. This contrasts with rearrangements of DUSP22, which are also observed in ALK- ALCLs and confer a good prognosis. The function and mechanisms of TP63 rearrangements and TP63 fusion proteins in tumorigenesis are poorly understood. As a result, attempts to treat these patients to date have been largely empiric. Thus, there is an urgent need to understand how TP63 fusions contribute to tumorigenesis and to translate the findings into novel therapeutic options for these patients. Here, we demonstrated that TP63 fusions are essential for the propagation of T-cell lymphomas (TCLs). Knockdown of TP63 fusions with specific shRNAs in TCL cell lines harboring TP63 rearrangements suppressed both cell growth in vitro and tumor growth in vivo. Retroviral expression of TBL1XR1-TP63, the most common TP63 fusion, conferred cytokine independence in Ba/F3 cells, consistent with its role as an oncogene. To investigate the role of TP63 fusions in T- and B-cell development and lymphomagenesis, we engineered a CAG-Loxp-Stop-Loxp-TBL1XR1-TP63 conditional knock-in mouse model and crossed with hCD2-Cre mice, which results in expression beginning during early lymphoid development. Transgenic mice had both cytokine and transcriptional evidence of immune activation as well as T cell repertoire narrowing, consistent with clonal selection. As observed in patients, transgenic mice developed multiple subtypes of both T- and B-cell lymphoma that mimicked ALCL, PTCL-NOS and MYC-expressing DLBCL. To define the effects and mechanisms of TP63 fusions within T cells, we performed CRISPR screening, transcriptomic, epigenomic, and proteomic analyses. We also performed a domain-focused CRISPR-Cas9 knockout scan using 452 sgRNAs tiling the entire coding regions of TBL1XR1 and TP63 to clarify essential oncogenic domains within TBL1XR1-TP63. Our data showed that domains within both the N-terminal TBL1XR1 and C-terminal TP63 portions contribute to the function of this fusion. We found that the N-terminal component of TP63 fusions (TBL1XR1, FOXK2, etc.) interacts with components of the NCOR/SMRT complex. At the same time, the C-terminal portion of TP63 fusions (which recapitulates the deltaNp63 isoform expressed in some carcinomas) interacts with the enhancer modifier KMT2D and its complex members. TBL1XR1-TP63 binds to a novel distal enhancer to drive MYC expression, and thus upregulates the expression of EZH2, which encodes the histone H3K27 methylase. Knockdown of the TP63 fusion in TCL cell lines led to complete loss of H3K27me3. Finally, we assessed whether EZH2 is a vulnerability of TP63-rearranged lymphomas. We found that knockdown of EZH2 in TP63-rearranged lines significantly impaired cell growth, as did treatment with the EZH2 inhibitor tazemetostat and the EZH2 and 1 dual inhibitor valemetostat. Valemetostat, which is now being tested in patients with lymphoma, was highly effective in three different in vivo models of TP63-rearranged TCL. Together, our results identify the TP63 fusion as a highly unique oncogenic driver in lymphomagenesis capable of recruiting multiple epigenetic modifier complexes and inducing a targetable dependence on EZH2.

HDAC8 Disrupts DNA Damage Response By Deacetylation of U2AF1 in Inv(16) Acute Myeloid Leukemia

Genomic instability due to defective DNA damage response (DDR) and DNA repair increases the probability of acquiring mutations, which may contribute to the pathogenesis of acute myeloid leukemia (AML). Inv(16) AML [inv(16)(p13q22) or t(16;16)(p13.1;q22)] creates a fusion gene CBFb-MYH11 (CM) by fusing core binding factor b (CBFb) with MYH11 which encodes smooth muscle myosin heavy chain (SMMHC). Our previous studies using a conditional Cbfb-MYH11 knock-in (KI) mouse model showed that preleukemic progenitors are prone to leukemia initiation, however, the underlying mechanism is not clear. Here, we show increased DNA double-strand breaks measured by comet assay and γH2AX staining in CM preleukemic progenitors compared to control counterparts. RNA-seq using sorted LSK (Lin-Sca1+Kit+) cells isolated from BM of CM KI mice (n = 5) and control (n = 4) identified a total of 1019 genes differentially expressed (526 up; 493 down; > 1.5-fold change; p < 0.01). Gene set enrichment analysis revealed that G2/M checkpoint, E2F targets, and mitotic spindle pathways were differentially downregulated in CM LSK, suggesting a possible contribution of cell cycle checkpoint dysregulation. To further explore the mechanism underlying CM-induced DNA damage, we expressed CM in 32D cells and subjected them to irradiation (IR; 3.5 Gy) followed by γH2AX and RAD51 staining. Significantly more γH2AX foci per nucleus were observed in CM cells (CM 35.03 vs Ctrl 24.71 at 0.5h, p<0.0001; CM 14.79 vs Ctrl 11.67 at 3h, p=0.0186), while fewer RAD51 foci per nucleus were seen in CM cells (CM 4.932 vs Ctrl 9.6 at 3h, p<0.0001), suggesting that CM may impair homologous recombination (HR)-mediated DNA repair. We reported that CM interacts with HDAC8 and enhances its activity, thus we examined whether HDAC8 may mediate the impact on DDR and HR activity. HDAC8 overexpression (OE) in 32D cells resulted in significantly more γH2AX foci per nucleus after IR (3.5 Gy) (HDAC8-OE 42.20 vs Ctrl 32.17 at 0.5h, p<0.0001; HDAC8-OE 15.45 vs Ctrl 10.63 at 3h, p<0.0001). Similar to CM cells, fewer RAD51 foci per nucleus were seen in HDAC8-OE cells (HDAC8-OE 11.63 vs Ctrl 17.39 at 3h, p<0.0001). To verify HDAC8 function in HR repair, we used the I-SceI and DR-GFP (homology-directed repair) or EJ5-GFP (end-joining-directed repair) DNA repair reporter (Gunn A et al, Methods Mol Biol, 2012). The frequency of repaired GFP+ population in the HR-directed repair reporter was reduced in HDAC8-OE cells compared to control (HDAC8-OE 7.807% vs Ctrl 11.34%, p=0.0169), while no difference was observed in the NHEJ-directed repair reporter. These results indicate that high HDAC8 expression disturbs HR DNA repair. The U2 small nuclear RNA auxiliary factor 1 (U2AF1) has been reported as a component of BRCA1-mRNA splicing machinery that regulates pre-mRNA splicing of critical genes involved in HR DNA repair. Here, we show that U2AF1 is subjected to post-translational acetylation and that U2AF1 interacts with HDAC8. We show that U2AF1 acetylation and its interaction with HDAC8 were increased following DNA damage and that HDAC8 is critical for deacetylating U2AF1. Site-directed mutagenesis for reported putative acetylation lysine sites (K15, K23, K175) (Elia AE et al, Mol Cell, 2015) revealed that the acetylation level of U2AF1-K23R decreased after IR and corresponded to reduced interaction with HDAC8 and the BRCA1/BCLAF1 complex. Notably, the interaction with BRCA1/BCLAF1 complex was rescued by mutating lysine to the acetylation mimic glutamine (Q). These results indicate that the acetylation state of U2AF1 is essential for the assembly of BRCA1-mRNA splicing complex and suggest that HDAC8 may regulate HR DNA repair by modulating the U2AF1 acetylation state. Similar to HDAC8 OE, CM expression reduced U2AF1 acetylation and impaired its interaction with BRCA1/BCLAF1 complex. Collectively, our results suggest that the increased DNA damage induced by CM may be due to the reduced acetylation of U2AF1 resulting from high HDAC8 activity. These results highlight a direct impact of inv(16) fusion protein and high HDAC8 activity on increased genomic instability in AML.