Background Loss-of-function TET2 mutations (TET2MT) occur in up to 30% of Myelodysplastic Syndrome and Acute Myeloid Leukemia. In Chronic Myelomonocytic Leukemia it is > 66%. Additionally, TET2MT is a founding lesion in myeloid neoplasms (MN), frequently appearing in clonal hematopoiesis of indeterminate potential (CHIP), a prodromal condition that increases the risk for MN and other diseases. Therefore, targeting the founder TET2MT clone represents a promising strategy to disrupt clonal proliferation at its source. We discovered that the loss of TET2 leads to metabolic rewiring, making cancer cells vulnerable to therapeutic interventions targeting glutamine metabolism (Gu et. al. Blood 2023). CB839, a potent glutaminase inhibitor, effectively disrupts glutaminolysis. However, previous clinical attempts to target glutamine metabolism pathways using CB839 have been unsuccessful. We propose that TET2MT creates unique metabolic signatures that can be specifically targeted with CB839. Consequently, TET2MT can serve as biomarkers for glutaminolysis inhibitors in the selective treatment of AML. Methods and Results Utilizing CRISPR-Cas9 edited clonal isogenic TET2WT and TET2KO cells derived from THP1 (Guan et al., 2021), coupled with HR LC-MS, we identified ~5000 features through untargeted analysis, with significant changes in 810. Targeted analysis confirmed unique metabolic disturbances in amino acid, glutamine, methionine, nicotinamide, and glycolytic pathways in TET2KO compared to TET2WT. Pathway enrichment analysis highlighted glutamine/glutamate biosynthesis as the most disturbed pathway. Significantly lower glutamine levels were observed in TET2WT patient cells and TET2KO mice compared to wildtype controls. These changes are primarily due to intracellular effects, as reflected in the significantly upregulated expression and activity of glutaminase (GLS1) and glutamate dehydrogenase (GULD1) in TET2KO cells. We conducted 13C glucose and 13C/15N glutamine metabolic flux analysis with complete carbon and nitrogen atom tracing of Glucose and Glutamine. Media glucose consumption was similar in TET2KO and TET2WT cells. However, TET2KO showed a significant buildup of cellular glucose and a slower consumption rate of glucose-6-phosphate, indicating slower glycolysis. In TET2WT cells, > 60% of αKG comes from glycolysis, while in TET2KO cells, it is < 40%, consistent with higher flux from Glutamine due to increased GLS1 and GULD1 activity (Gu et al., Blood 2023). Our data suggest that TET2MT creates metabolic reprogramming, leading to heightened glutamine addiction that can be therapeutically exploited. Consistent with our hypothesis, loss of TET2 resulted in high sensitivity to glutamine restriction and treatment with the GLS1 inhibitor CB839 (Jiang, B., et al. Mol Cell 2022). We performed metabolic analysis to understand how CB839 limits cell growth in TET2KO cells. The most impacted pathway was glutamine metabolism in the mitochondria, where glutamine replenishes the TCA cycle. Glutamic acid levels were reduced more than two-fold with CB839 treatment in TET2KO compared to TET2WT. As a result, TET2KO cells showed a 3-5-fold reduction in αKG with CB839 treatment compared to TET2WT. Significant reductions in αKG downstream TCA cycle compounds, such as succinate, fumarate, and malate, were observed, with less impact on upstream compounds citrate and oxaloacetate. Inhibition of glutaminase reduced overall downstream metabolites, while IMP, the first product of de novo purine synthesis, increased 2-3-fold. These findings confirm that TET2 loss promotes glutamine metabolic pathway addiction, with CB839 inhibiting TET2KO cell growth by disrupting the glutamine-dependent TCA cycle. Given that the TET2 inhibitors Eltrombopag (Epag) and TETi76 mimic the loss of TET2 and create similar disruptions in glutaminolysis, a combination therapy approach for non-TET2 mutated AML using TETi and CB839 can be utilized. Conclusion. Our study provides a detailed mechanistic understanding of how TET2 mutations contribute to MN and CHIP. This discovery paves the way for a biomarker-driven, novel therapeutic strategy for myeloid leukemia. Furthermore, our research highlights the potential of combining TET2 inhibitors with glutaminolysis inhibitors as a pioneering treatment approach for a broad range of leukemia types.

Background: One of the major challenges in the treatment of acute myeloid leukemia (AML) is the elimination of undifferentiated immature blood cells, often referred to as leukemia stem and progenitor cells (LSPC). LSPCs persist after treatment and are considered a major cause of relapse and refractory disease. Despite initially high remission rates with recent advances in treatment strategies, relapse occurs in a large proportion of patients, leading to high mortality in AML. Myeloid neoplasia (MN) is driven by somatic mutations, with loss of function TET2 mutations (TET2MT) being one of the most common somatic lesions in MN. TET2, a member of the TET-family of DNA dioxygenases, is the major methyl-cytosine oxidase in hematopoietic cells, including T-cells. TET2 (along with TET1/3) are Fe2+ and αKG-dependent dioxygenases that utilize molecular oxygen to progressively oxidize 5-methyl cytosine (mC) in promoters, enhancers and silencer-associated mCpG segments of the genome, leading to their demethylation. This process is essential for mounting an accurate and efficient gene transcription profile that determines cell lineage fate, differentiation and proliferation. Here we report, while loss of TET2 creates a proliferative advantage to the LSPCs, it may also make them immunogenic and susceptible to immune surveillance due to higher expression of MHC class I/II molecules. Methods: We generated isogenic TET2 knockout (TET2KO) leukemic cell models in THP1 and K562 cell lines using CRISPR-Cas9 gene editing. We also utilized natural TET2-deficient cell models, including OCI-AML5 (TET2+/-) and SIMG5 (TET2-/-). Western blot (WB) analysis, bicolor immunofluorescent (IF) imaging and confocal microscope were employed to assess protein expression and protein subcellular localization in these models. To validate our findings, we performed flow cytometry. In addition, we analyzed the HLA expression in TET2MT mutant associated MN patients and compared it with WT. Results: Our study revealed that HLA- A/-B/-C (pan-HLA) expression in THP1 cells (MHC Class I positive) increased by more than 2-fold as a consequence of TET2 deletion compared to their isogenic wildtype counterparts, as observed in both WB and IF analyses. Additionally, pan-HLA protein levels were also significantly higher in cells with naturally TET2 deficiency (OCI-AML5 and SIGM5) compared to TET2 wildtype cells (THP1). The increased HLA protein levels was further confirmed by treatment with small molecule inhibitor of TET2 (TETi). Our data demonstrated that TETi treatment significantly increased pan-HLA expression in TET2WT cells but not in TET2KO cells. Furthermore, treatment with interferon gamma (INFg), a known inducer for HLA expression, significantly increased pan-HLA protein levels in TET2WT but not in TET2KO, suggesting that HLA expression is epigenetically regulated by TET2 in interferon mechanism of action. Interestingly, K562, an erythroid leukemia cell line known to be negative for surface expression of HLA- A/-B/-C had a significant pan-HLA upregulation upon genetic deletion or small molecule inhibition of TET2 dioxygenase activity. This observation was further confirmed in our analysis of human myeloid neoplasia (MDS and AML) patient cohort. Analysis of the RNAseq data from a cohort of 157 MN patients, including 38 with TET2MT and 64 healthy bone marrow samples, demonstrated that both MHC class I and II were upregulated in TET2 mutant MN patients. Conclusion: Our findings demonstrate that the loss of TET2 function significantly increased the surface expression of MHC class I/II proteins in both isogenic and natural TET2-deficient leukemic cell models, as well as in MDS and AML patients, compared to TET2WT and healthy control. The use of TET family inhibitors to chemically mimic TET2 loss further substantiated the increased pan-HLA expression, specifically in TET2 wildtype cells. Moreover, the differential response to INFγ treatment between TET2 wildtype and TET2 knockout cells highlights the critical role of TET2 in regulating HLA protein expression. These results suggest that targeting TET2 could enhance anti-cancer immune responses by increasing MHC class I/II expression, thereby improving the efficacy of immunotherapy therapies. This study provides a promising avenue for advancing cancer immunotherapy through epigenetic modulation.

Iron protoporphyrin IX (heme) is a redox-active cofactor that is bound in mammalian cells by GAPDH and allocated by a process influenced by physiologic levels of NO. This impacts the activity of many heme proteins including indoleamine dioxygenase-1 (IDO1), a redox enzyme involved in immune response and tumor growth. To gain further understanding we created a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after labeling could indicate its heme binding by fluorescence quenching. When purified or expressed in a human cell line, TC-hGAPDH had properties like native GAPDH and heme binding quenched its fluorescence by 45–65%, allowing it to report on GAPDH binding of mitochondrially-generated heme in live cells in real time. In cells with active mitochondrial heme synthesis, low-level NO exposure increased heme allocation to IDO1 while keeping the TC-hGAPDH heme level constant due to replenishment by mitochondria. When mitochondrial heme synthesis was blocked, low NO caused a near complete transfer of the existing heme in TC-hGAPDH to IDO1 in a process that required IDO1 be able to bind the heme and have an active hsp90 present. Higher NO exposure had the opposite effect and caused IDO1 heme to transfer back to TC-hGAPDH. This demonstrated: (i) flow of mitochondrial heme through GAPDH is tightly coupled to target delivery, (ii) NO up- or down-regulates IDO1 activity by promoting a conserved heme exchange with GAPDH that goes in either direction according to the NO exposure level. The ability to drive a concentration-dependent, reversible protein heme exchange is unprecedented and reveals a new role for NO in biology.

Amyloidosis, also known as light chain amyloidosis (AL), is one of the most common types of systemic amyloidosis affecting patients of older ages with a median age of 64 years. Nearly 4000 new cases are diagnosed annually in the US alone (Dima et. al. JCO Oncology Practice 2023). AL is a proteostasis disorder stemming from misfolded immunoglobulin light chains caused by abnormal protein production by clonal plasma cells and in rare cases clonal B cells. These misfolded proteins can be deposited in the organs, tissues, and nerves and lead to permanent and even lethal damage. This dysregulated feature of AL resembles findings in multiple myeloma (MM) but there are several differences in the pathogenesis (Madan et. al. Mayo Clinic Proceedings 2010). Due to the lack of molecular biomarkers for detection at early stages, AL is usually diagnosed at a late stage when significant organ failure is already present. There is a high rate of hospitalization due to heart failure, renal failure, and other debilitating symptoms (Dima et. al. JCO Oncology Practice 2023). Thus, there is an urgent need to understand the molecular pathogenesis of AL and to develop biomarkers and therapeutic strategies to prevent clonal evolution. Although AL and MM are clinically distinct, there are several shared clinical features with overlapping pathogenesis (Madan et. al. Mayo Clinic Proceedings 2010). The mechanisms that confer amyloidogenicity on clonal plasma cells have yet to be fully elucidated. Our aim is to achieve an improved and novel understanding of the distinct transcriptional programs that confer amyloidogenicity to plasma cells. For this purpose, we have adopted a unique approach utilizing next-generation sequencing of the CD138+ and CD138- fraction of a large cohort of AL patients. We will report the transcriptional profile of AL amyloid plasma cell clones and the cellular microenvironment they interact with to identify molecular biomarkers and potential therapeutic targets. In this study, we used treatment-naïve human frozen banked plasma cells and sorted bone marrow aspirate samples of AL patients. The 67 out of 84 patient samples yielded a good quality of RNA, DNA, and proteins. Among 67 samples, 22 were AL, 31 confirmed multiple myeloma (MM), 3 overlapped with MM/AL, 2 were smoldering myeloma (sMM), and 9 were monoclonal gammopathy of undetermined significance (MGUS). Clinical annotation was obtained regarding bone marrow clonal burden, treatment history, and response. We used analytical flow cytometry coupled with the purification of plasma cells using antibodies against CD138 (MicroBeads Kit). The percentages of CD138+ fraction ranged from 0.04% to 14.1%. We utilized samples with 5% or more (n=10) to purify CD138+ and CD138- cells. These ten samples include AL (5 cases), MGUS (3 cases), MM (1 case), and sMM (1 case). RNA from CD138+ cells as well as CD138- cells of these samples along with bulk RNA from all 67 samples were extracted and next-generation RNA sequencing was performed. The RNASeq data were analyzed to identify the novel and unique clonal AL amyloidosis profile compared to MM and normal cells. In addition, to compare the proteostasis pathways of AL and MM, we utilized four distinct cellular modes of MM (MM1.S, RPMI8226, KMS12, and U266). Our preliminary data demonstrated gain in endoplasmic reticulum (ER) function as one of the common mechanisms in AL and MM. Consistent with our previous report about MM, (Hasipek et. al. Cancers 2021) we observed that therapeutic resistance and persistence of plasma cell clones emerge as a result of gain in the function of ER, unfolded protein response, and ER-dependent protein degradation. To further understand the mechanism of AL, the expression level of PDIA1 (protein disulfide isomerase A1) was investigated. PDIA1, a resident ER chaperon essential for light chain exit from plasma cells, is a major member of the protein disulphide isomerase family involved in the protein folding process. Inhibition of PDIA1 positively correlates with the survival rate of patients with relapsed myeloma (Hasipek et. al. Cancers 2021). The levels of PDIA1 in 67 samples were compared to identify PDIA1 as a potential therapeutic target in both AL and MM. In summary, this study establishes the basis of the novel predictive model for AL in both molecular pathogenesis and transcriptional profile with the potential to develop novel biomarkers and therapeutic targets.

Background: Antibodies directed against leukemia-specific antigens have been an early form of immunotherapy. Chimeric antigen receptor T-cell therapy (CART) has shown tremendous success in lymphoid neoplasia, but both CART cells and antibodies are limited due to their restriction to cell surface-expressed proteins. In contrast, T cell responses recognizing leukemia cells via tumor-specific peptides have a broader spectrum of targets and are believed to mount the strongest tumor surveillance and anti-tumor response. One major roadblock in developing effective immune therapies is the inability to identify tumor cell-specific neoantigens that can distinguish normal cells from cancer cells. Neoantigens can induce anti-tumor effects through inducible T-cell responses, thereby activating cellular immunotherapy with the potential to eradicate Acute Myeloid Leukemia (AML). Selected studies have shown that neoantigens encoded by recurrent genetic aberrations in AML can be targeted using immunotherapy. Loss of function TET2 mutations (TET2MT) have been frequently identified in myeloid neoplasia (MN), contributing to disease pathogenesis and clonal expansion as indicated by high prevalence of TET2MT in clonal hematopoiesis of indeterminate potential (CHIP). Targeting TET2MT in AML may not only effectively eradicate leukemia, but also pre-malignant hematopoietic stem cells, thereby disrupt clonal proliferation at its source. Here we report the identification of neoantigens associated with TET2MT that will help to develop novel therapeutic strategy. Methods: We utilized CRSPR-Cas9 edited clonal isogenic TET2WT and TET2KO cells derived from THP1 (Guan et al., Blood Cancer Discov, 2021). Three independent biological replicates of TET2ko and TET2WT were used for immunoaffinity purification of cell surface HLA bound peptides. The solubilized HLA complexes were dissociated in acid and filtered through 5kDa NMWCO ultrafiltration to remove non-peptide small molecules. Resulting peptide fractions were analyzed on an Orbitrap mass spectrometer. The raw data files were searched with Protein discovery with 5% FDR and peptide with 7-15 amino acids in length. HLA-A binding peptides were identified by mapping peptide FASTA sequences and HLA class alleles in the Net MHC pan 4.1 database. Results: Through optimized immunopeptidome workflow, we found that the amount of antibody used in immunoprecipitation was critical for maximum enrichment of immunopeptides. Over 5000 HLA-A binding peptides were enriched for quality analysis. From all identified peptides, about 30% were filtered out as non-immunopeptides due to their lengths not being within 7-15 amino acids and derivation from most abundant cellular proteins. The majority of identified peptides were 9mers. We used unsupervised alignment and clustering of peptide sequences based on HLA-A binding motifs. About 98% of the eluted peptides clustered into five motifs with high affinities for HLA-A binding, typically featuring leucine (L) at positions 2 and 9. We also calculated the immunogenicity of eluted peptides in silico using the Immune Epitope Database (IEDB) based on T-cell preferences for amino acids. 21.8% of eluted peptides had an immunogenicity score above 0.2, suggesting they are likely T-cell epitopes. Protein pathway analysis revealed the top contributing functional groups, including antigen processing, separation of sister chromatids, cell cycle checkpoints, and DNA repair. TET2 deficiency created a distinct immunopeptidome, with about threefold more eluted peptides identified in isogenic TETKO compared to TET2WT, suggesting higher possible T-cell immunogenicity. A total of 389 peptides were either exclusively identified in TETKO cells or exhibited a 2-fold higher mass intensity compared to TET2WT. From these enriched in TETKO cells, 10 peptides were selected based on their higher immunogenicity and signal intensity for further evaluation as potential targets for immunotherapeutic approaches. Conclusions: Our study revealed that TET2 loss leads to a unique peptide repertoire, suggesting enhanced T-cell immunogenicity. Our findings offer promising avenues for the development of novel diagnostic tools and therapeutic strategies targeting TET2-associated neoantigens in AML, with the potential to improve patient outcomes by reducing the risk of relapse and eradicating both malignant and pre-malignant cells.

Circulating myeloid cells carrying somatic mutations characteristic of myeloid neoplasms (MN) are also found in a significant proportion of otherwise healthy, mostly elderly individuals in a prodromal condition called clonal hematopoiesis of indeterminate potential (CHIP). CHIP carriers have an increased risk (10-25x) of MN, including treatment-related MN in cancer survivors who received chemotherapy and/or ionizing radiation. These individuals also face an increased risk of cardiovascular disease and pulmonary atrial hypotrophy (PAH), among other diseases. However, there are currently no known therapeutic options to address CHIP. Recent reports suggest that the age-related inflammatory microenvironment profoundly affect the evolution of CHIP (Guarnera L & Jha BK. Semin Hematol. 2024). TET2 mutation (TET2MT) is the second most frequent somatic lesion in CHIP. The loss of TET2 results in an aberrant pattern of 5-methyl cytosine (mC), creating a volatile pre-neoplastic state in hematopoietic stem and progenitor cells (HSPCs). Clonal evolution of TET2MT HSPCs is strongly age dependent. TET2MT often occurs as a founding lesion, implying that eliminating the TET2MT clone would impact CHIP at an early ontogenetic stage. We assessed two distinct strategies to target TET2-deficient clones: (i) a synthetic lethal approach, where the TET2MT clone can be targeted by blocking residual activity (Guan et al., Blood Cancer Discov. 2021) while simultaneously increasing the fitness of TET2 proficient normal HSPCs and (ii) an anti-inflammatory regimen, where given that TET2MT clone creates and persists in pro-inflammatory milieu, targeting inflammatory pathways may prevent clonal evolution. In this study, we screened three nontoxic and well tolerated anti-inflammatory molecules (Nicotinamide riboside (NR), Danazol, and Ibuprofen) along with TETi76, a known TET inhibitor that restricts the evolution of TET2MT by inhibiting residual TET-dioxygenase activity. Here, we report two complimentary low regimen strategies for restricting TET2MT CHIP in vivo. We developed Tet2-/- B6 CD45.2 TomatoRed mTmG murine models to facilitate easy monitoring of the Tet2mt graft. Competitive bone marrow transplant coupled with spectral flow cytometry was used to evaluate clonal burden and its impact on polyclonal normal hematopoiesis. Each graft (2x106 cells; 10% Tet2-/-TomatoRed+ 90% WT Bl6CD45.2) were transplanted into JaxBoy CD45.1 recipients. Three weeks post-transplant, mice were randomized into five treatment groups (n=8/group): Vehicle, TETi76 (25mg/kg), NR (300mg/kg), Ibuprofen (40mg/kg), or Danazol (35mg/kg). Peripheral blood was analyzed by flow at 3, 6, 24, and 52 weeks using CD45.1, CD45.2, and Tomato to assess CHIP expansion. Mice were sacrificed at 52 weeks and analysis of blood, bone marrow, and spleen were performed by spectral flow cytometry. At the time of sacrifice, the Tet2-/- fractions traced by the TomatoRed were as follows: 75% (vehicle), 43% (TETi76, p= 0.003), 61% (NR, p=0.054), 62% (ibuprofen p=0.21) and 48% (Danazol p=0.0187). The mean of Tet2-/- fraction in the control group was slightly higher than in the groups treated with anti-inflammatory drugs. Our data indicate that synthetic lethality induced by TETi76 restricted the evolution of Tet2-/- HSPCs and simultaneously restored normal hematopoiesis. The anti-inflammatory treatments moderately slowed Tet2-/- CHIP expansion. Among the other drugs used, Danazol showed a significant reduction in overall Tet2-/- fraction, while ibuprofen had no significant effect. HPSC differentiation analysis showed that CD11b+ populations were decreased in mice treated with TETi76 (3-fold; p =0.054), with moderate effects observed with (<1.5-fold) compared to vehicle control. Interestingly, treatments mitigated the myeloid skewing of Tet2-/- fraction reflected in an increased CD3+ population (5-fold; p=0.003) with milder effects observed for Danazol (<2-fold, p=0.18), NR (<2 fold; p= 0.41), and Ibuprofen (3-fold, p=0.0469) compared to the vehicle control. In summary, treatment with TETi76 slowed the expansion of Tet2mt CHIP in vivo. The effects of anti-inflammatory drugs were moderate. A complete to partial restoration of normal hematopoiesis was observed by TETi76. Our results indicated this low regimen, non-toxic treatment could be a potential option for preventing TET2MT CHIP.

Background: Methyl-CpG binding domain 4 (MBD4) is a known DNA glycosylase involved in critical steps of base excision repair (BER) and DNA mismatch repair (MMR) and is essential for reducing genotoxic stress in normal cells. MBD4 binds to 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) - oxidation products of TET2 dioxygenases - and may initiate a multi-step BER/MMR around mCpG/hmCpG DNA. Loss of function (LOF) mutations in MBD4 (MBD4 MT) result in compromised BER/MMR, leading to genomic instability. Certain germline MBD4 variants have been reported to be predisposing factors for early onset AML, among other cancers. 1,2 In cases screened at our institution, biallelic MBD4 p.R431* & p.L563* tracked with AML in brother and sister. 3 This inspired us to hypothesize that partial or complete loss of MBD4 may result in reduced BER/MMR function and ultimately in genomic instability and faster accumulation of somatic lesions favoring malignant evolution. Methods: We performed NGS and epigenetic studies coupled with detailed biochemical analyses in CRISPR engineered cell lines and primary AML cells and in vivo model systems to dissect the mechanism of clonal evolution of MBD4 MT hematopoietic stem and progenitor cells (HSPCs). Results: We analyzed WES data from 1,692 patients with myeloid neoplasms (MN, 732 MDS; 960 AML). After adequate filtering based on frequency in healthy population (odds ratio of >1.5), 16 variants in MBD4 were identified in 24 MN cases (MDS=9, AML=15). Among them, 4 heterozygous variants of unknown significance of suspected germline nature (p.R425Q, p.N461S, p.R486K, p.D504H) mapped in the glycosylase domain. Comparing MBD4 MT vs. wild-type ( MBD4 WT) cases, MBD4 MT were found in younger patients (median 55 vs. 71 yrs; p = 0.012) with TET2 MT co-occurring at a lower frequency (8 vs. 24%). Since MBD4 is known to preferentially bind with 5hmCpG DNA, a stable epigenetic mark induced by TET2, we investigated the biochemical basis of MBD4 dysfunction and its connection to LOF TET2 MT. We show that MBD4 hydrolase activity levels affect the genomic accumulation of 5hmC in a TET2-dependent manner. MBD4 KO led to more than 2-fold increased 5hmC in TET2WT cells but not in TET2KO cells, suggesting a functional connection between MBD4 and TET2 in BER/MMR. To test the function and effects of the 4 variants (p.R425Q, p.N461S, p.R486K, p.D504H) identified in our cohort on the activity and function of MBD4, we generated these mutations in a pcDNA plasmid using site directed mutagenesis. The mutants were ectopically expressed in isogenic CRISPR-engineered MBD4 KO HEK293 cells and its effects on 5hmC accumulation were tested. The impact on the functional outcomes were compared to WT controls as described earlier. 4 We found that these 4 variants lead to MBD4 protein instability, and thereby lower MBD4 DNA glycosylase activity resulting in increased (1.6 to 3.3-fold) genomic accumulation of TET2-dependent oxidation products. To establish the effect of MBD4 MT in primary cells, we generated CRISPR-engineered human CD34 + HSPCs and assessed their clonal evolution via colony forming assay. Loss of MBD4 led to clonal advantage reflected in persistent colonies in MBD4KO CD34 + HSPCs that demonstrated myeloid differentiation bias similar to what was observed in patients, suggesting that MBD4 MT may favor clonal evolution to fully blown MN. Finally, transcriptomics analysis of MBD4KO in K562 cells in the presence and absence of functional TET2 demonstrated loss of BER genes compared to WT. The co-immunoprecipitation of MBD4 followed by western blot analysis identified XRCC1, a key component of the BER complex, as a part of MBD4 interactome. Comprehensive characterization of protein complexes with LCMS/MS in K562 cells suggests that MBD4 is involved in maintaining genomic integrity. Consistent with our hypothesis, MBD4 KO in K562 and THP1 cells resulted in a 2- and 3-fold increase in spontaneous mutations respectively, as observed via HPRT assay. Conclusions: Genomic instability due to MMR/BER dysfunction in MBD4 MT cells may result in a higher rate of clonal progression. Interestingly, MBD4 deficiency may provide opportunity for therapeutic targeting of DNA repair pathways in a subset of MBD4 MT-associated MN. Indeed, our findings indicate the possibility of leveraging MBD4 as an actionable target to regulate DNA methylation in association with TET2 during leukemogenesis.

Background. TET2 mutations ( TET2 MT) are the most frequently observed somatic lesions in myeloid neoplasms (MN). TET2 belongs to an Fe2+ and α-ketoglutarate (αKG) dependent DNA-dioxygenase family that progressively oxidizes 5-methyldeoxycytidine (5mC), leading to promoter and enhancer mCpG demethylation essential for efficient transcription. TET2 accounts for the majority of DNA dioxygenase activity in hematopoietic stem and progenitor cells (HSPCs), and following its loss, the resulting accumulation of 5mC leads to a loss of lineage plasticity and transcriptional repression of tumor suppressor genes, thereby establishing a volatile pre-neoplastic state. TET2 is one of the major consumers of αKG, a major cofactor produced by the tricarboxylic acid (TCA) cycle. Therefore, loss of TET2 creates a metabolic perturbation in HSPCs. However, the impact of TET2 loss on the metabolism of leukemic cells and its subsequent effects on malignant evolution remains unclear. Methods. We generated isogenic TET2 -/- ( TET2 ko) leukemic cell models in THP1 and K562 using CRISPR-Cas9 knockout. We then utilized targeted and untargeted metabolic analyses by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry in these models. We aimed to investigate the intricate metabolic changes associated with TET2 and TET2 MT. To validate our findings, we extended our studies to primary cells derived from MN patients with and without TET2 lesions. We performed extensive cellular metabolism studies using 13C isotope labelled glucose and glutamine. Additionally, levels of 5mC and its oxidation products, namely 5-hydroxymethyldeoxycytidine, 5-formyldeoxycytidine, and 5-carboxydeoxycytidine were documented for correlating epigenetic modifications with TET2 activities. Results. Targeted metabolic analysis uncovered profound impacts of TET2 MT on key metabolic pathways including the TCA cycle, glycolysis, glutamine metabolism, and nucleoside biosynthesis (Figure A). Distinct changes were observed in cells from different lineage backgrounds, suggesting diverse metabolic pathway rewiring specific to cell origin. Despite the divergence among lineages, loss of TET2 created common and unique metabolic signatures, highlighting their crucial role in sustaining the survival and clonal advantages specific to TET2 MT. Representative signature compounds and their relative quantities in TET2 wildtype (WT) and TET2 MT are depicted in Figure A. The targeted metabolic analysis revealed a significantly lower level of glutamine in TET2 MTcells, suggesting a potential preference for exhausting glutamine as a source of energy production. For validation, we conducted 13C metabolic flux analysis (13C-MFA) using glucose (U-13C, 99%) and L-glutamine (U-13C, 99%). 13C-MFA revealed distinct enrichment patterns (Figure B). In the TCA cycle, the dominant isotopologues receiving [U-13C]-glucose were M+2 for citrate and isocitrate. However, for aKG, succinate (SA) and fumarate (FA), the M+2s were significantly lower in TET2 -/- compared to WT, suggesting that the major source for these metabolites were rewired from glycolysis to glutamine metabolism in TET2 -/-. This was confirmed by [U-13C]-glutamine labeling where dominant isotopologues, M+5 for aKG and M+4 for SA, FA and malate, were significantly higher at 24-hour time point in in TET2 -/- compared to WT. Untargeted metabolic analyses from isogenic cells were used to identify metabolic features associated with TET2 loss. These metabolic features were also assayed in primary AML cells with TET2 MT (n=12) and without: RUNX1 (n=20), SF3B1 (n=11), IDH1 (n=10), IDH2 (n=13), FH (n=3), as well as healthy donors as controls (n=6). Based on the molecular features identified in the TET2 ko isogenic cells, MN patients with TET2, IDH1/2, and germline FH mutations clustered more closely, suggesting shared metabolic signatures among these groups. In contrast, patients with RUNX1 and SF3B1 mutations formed separate clusters, demonstrating distinct metabolic profiles associated with these genetic alterations. Conclusion. Our findings provide comprehensive and novel insights into the metabolic consequences of TET2 inactivation in MN. The identified metabolic signatures and rewiring of key pathways may hold promise as potential therapeutic targets and offer opportunities for tailored treatment approaches in TET2MT MN.

Introduction. Accumulation of undifferentiated immature blood cells is a hallmark of myeloid neoplasia (MN), and indicates decoupling of terminal-differentiation from proliferation. Therefore, reinstating to a programmed state of differentiation (lineage-maturation) is a treatment approach, exemplified by all-trans retinoic acid (ATRA) treatment of acute promyelocytic leukemias that contain t(15;17) (q24;q21). This translocation inactivates retinoic acid receptor alpha (RARA) due to its fusion with promyelocytic leukemia PML resulting in PML-RARA. This fusion gene responsible for cellular transformation confers sensitivity to treatment with ATRA. However, ATRA's therapeutic-success in APLs has not extended to other AML phenotypes. We and others have shown that RA is a ligand, not just for RARs, but also pro-survival peroxisome proliferator-activated receptor β/δ (PPARβ/δ), a member of the nuclear hormone receptor superfamily transcription factors that regulate several metabolic pathways. RA is transported by two carrier proteins, cellular retinoid-binding protein CRABP2, or fatty-acid-binding protein 5 (FABP5), depending on cellular context. These carriers preferentially deliver RA to RARs versus PPARβ/δ respectively. Somatic lesions such as loss of function TET2 or Tp53 mutation, NPM1 and ASXL1 mutations, are associated with upregulated FABP5 and downregulated CRABP2. In AML cells with these mutations, therefore, the high FABP5 can be expected to deliver RA to PPARβ/δ instead of RAR, to potentially contribute to decoupling of differentiation from proliferation. We therefore investigated this possibility and its treatment implications. Methods. Overexpression, gene knockdown/knockout (ko/kd) of key regulators of RAR and PPARβ/δ were generated in specific genotypes of AMLs. Small molecule inhibitors of FABP5 were used to further interrogate the pathways in a translationally relevant manner, both in vitro, in both malignant and normal cells, and in pre-clinical in vivo models. Results. Gene expression analysis of AMLs in the Beat AML and TCGA databases demonstrated upregulation of pro-survival PPARβ/δ target genes including FABP5 and downregulation of differentiation promoting RAR pathway genes including CRABP2in AMLs 1. In particular, FABP5 was highly expressed in AMLs containing TET2 NPM1, TP53 and ASXL1 mutations. AML cell lines OCIAML3 (NPM1 ins/DNMT3a R882c), OCIAML5 (ASXL1 Y591*/TET2 S825*), THP1 (TP53 del) and isogenic CRISPR engineered K562 (TET2 +/+/TET2 -/-) demonstrated 4-100 fold higher FABP5 expression compared to normal myeloid precursors. These models did not differentiate in response to pharmacologic concentrations of ATRA. To test if FABP5 is necessary and sufficient to maintain the undifferentiated state of these cells in the presence of ATRA, we genetically inactivated FABP5 either using RNAi (kd) or CRISPR (ko). FABP5 knockdown or knockout cells created sensitivity to ATRA, resulting in terminal-differentiation as observed by flow cytometry for terminal-differentiation markers ( See Figure). FABP5 was then inhibited using highly specific small molecule FABP5 inhibitors with a 56-fold higher affinity compared to ATRA 1: combination of iFABP5 and ATRA induced terminal-differentiation with significant upregulation of the monocyte lineage markers CD14 and granulocyte lineage marker CD11b measured by flow cytometry at 72h. Toinvestigatewhether knocking down FABP5 and ATRA treatment affects gene expression changes we performed transcriptomic profiling of OCIMAL3 Scr, shFABP5 cells treated with or without ATRA using whole exome RNA sequencing. Treatment of FABP5 kd OCIMAL3 cells with ATRA resulted in the upregulation of RAR pathways genes and downregulation of proliferation promoting genes. Conclusion. In sum, recoupling of lineage-maturation to proliferation can be achieved in non-APL AMLs that highly express the RA carrier FABP5, by inhibiting FABP5 using small molecules. This candidate treatment modality is distinct from cytotoxic chemotherapy, and importantly, can spare normal hematopoiesis.

Nitric oxide (NO) is a ubiquitous cell signaling molecule which mediates widespread and diverse processes in the cell. These NO dependent effects often involve activation (e.g. NO binding to the heme group of soluble guanylyl cyclase for cGMP production) or inactivation (e.g. S-nitrosation) of protein targets. We studied the effect of NO and heme-NO on the transmembrane signaling enzyme NADPH oxidase 5 (NOX5), a heme protein which produces superoxide in response to increases in intracellular calcium. We found that treatment with NO donors increases NOX5 activity through heme-dependent effects, and that this effect could be recapitulated by the addition of heme-NO. This work adds to our understanding of NOX5 regulation in the cell but also provides a framework for understanding how NO could cause widespread changes in hemeprotein activity based on different affinities for heme v. heme-NO, and helps explain the opposing roles NO plays in activation and inactivation of hemeprotein targets.

NADPH oxidase 5 (NOX5) is a transmembrane signaling enzyme that produces superoxide in response to elevated cytosolic calcium. In addition to its association with numerous human diseases, NOX5 has recently been discovered to play crucial roles in the immune response and cardiovascular system. Details of NOX5 maturation, and specifically its response to changes in intracellular heme levels have remained unclear. Here we establish an experimental system in mammalian cells that allows us to probe the influence of heme availability on ROS production by NOX5. We identified a mode of dynamic regulatory control over NOX5 activity through modulation of its heme saturation and oligomeric state by intracellular heme levels and Hsp90 binding. This regulatory mechanism allows for fine-tuning and reversible modulation of NOX5 activity in response to stimuli.

Soluble guanylyl cyclase (sGC) is a key component of NO-cGMP signaling in mammals. Although heme must bind in the sGC β1 subunit (sGCβ) for sGC to function, how heme is delivered to sGCβ remains unknown. Given that GAPDH displays properties of a heme chaperone for inducible NO synthase, here we investigated whether heme delivery to apo-sGCβ involves GAPDH. We utilized an sGCβ reporter construct, tetra-Cys sGCβ, whose heme insertion can be followed by fluorescence quenching in live cells, assessed how lowering cell GAPDH expression impacts heme delivery, and examined whether expressing WT GAPDH or a GAPDH variant defective in heme binding recovers heme delivery. We also studied interaction between GAPDH and sGCβ in cells and their complex formation and potential heme transfer using purified proteins. We found that heme delivery to apo-sGCβ correlates with cellular GAPDH expression levels and depends on the ability of GAPDH to bind intracellular heme, that apo-sGCβ associates with GAPDH in cells and dissociates when heme binds sGCβ, and that the purified GAPDH-heme complex binds to apo-sGCβ and transfers its heme to sGCβ. On the basis of these results, we propose a model where GAPDH obtains mitochondrial heme and then forms a complex with apo-sGCβ to accomplish heme delivery to sGCβ. Our findings illuminate a critical step in sGC maturation and uncover an additional mechanism that regulates its activity in health and disease.

The enzyme soluble guanylyl cyclase (sGC) is a heterodimer composed of an α subunit and a heme-containing β subunit. It participates in signaling by generating cGMP in response to nitric oxide (NO). Heme insertion into the β1 subunit of sGC (sGCβ) is critical for function, and heat shock protein 90 (HSP90) associates with heme-free sGCβ (apo-sGCβ) to drive its heme insertion. Here, we tested the accuracy and relevance of a modeled apo-sGCβ–HSP90 complex by constructing sGCβ variants predicted to have an impaired interaction with HSP90. Using site-directed mutagenesis, purified recombinant proteins, mammalian cell expression, and fluorescence approaches, we found that (i) three regions in apo-sGCβ predicted by the model mediate direct complex formation with HSP90 both in vitro and in mammalian cells; (ii) such HSP90 complex formation directly correlates with the extent of heme insertion into apo-sGCβ and with cyclase activity; and (iii) apo-sGCβ mutants possessing an HSP90-binding defect instead bind to sGCα in cells and form inactive, heme-free sGC heterodimers. Our findings uncover the molecular features of the cellular apo-sGCβ–HSP90 complex and reveal its dual importance in enabling heme insertion while preventing inactive heterodimer formation during sGC maturation.

Nuclear receptors play an important role in prostate cancer and the androgen receptor is a key transcription factor in regulation of cellular events. Androgen receptor-associated coregulators may be upregulated or downregulated in prostate cancer. Altered expression of regulators may potentiate androgen-induced proliferation, migration, and invasion. Therapies aimed to modulate the function of coregulators in prostate cancer may be based on the use of small molecule inhibitors. Expression and function of AR-associated proteins could be investigated after overexpression and gene silencing followed by hormonal treatment, real-time RT-PCR and ChIP.

Standard treatment for metastatic prostate cancer (CaP) prevents ligand-activation of androgen receptor (AR). Despite initial remission, CaP progresses while relying on AR. AR transcriptional output controls CaP behavior and is an alternative therapeutic target, but its molecular regulation is poorly understood. Here, we show that action of activated AR partitions into fractions that are controlled preferentially by different coregulators. In a 452-AR-target gene panel, each of 18 clinically relevant coregulators mediates androgen-responsiveness of 0–57% genes and acts as a coactivator or corepressor in a gene-specific manner. Selectivity in coregulator-dependent AR action is reflected in differential AR binding site composition and involvement with CaP biology and progression. Isolation of a novel transcriptional mechanism in which WDR77 unites the actions of AR and p53, the major genomic drivers of lethal CaP, to control cell cycle progression provides proof-of-principle for treatment via selective interference with AR action by exploiting AR dependence on coregulators.

Androgen receptor (AR) is a ligand-activated transcription factor that is the main target for treatment of non-organ-confined prostate cancer (CaP). Failure of life-prolonging AR-targeting androgen deprivation therapy is due to flexibility in steroidogenic pathways that control intracrine androgen levels and variability in the AR transcriptional output. Androgen biosynthesis enzymes, androgen transporters and AR-associated coregulators are attractive novel CaP treatment targets. These proteins, however, are characterized by multiple transcript variants and isoforms, are subject to genomic alterations, and are differentially expressed among CaPs. Determining their therapeutic potential requires evaluation of extensive, diverse datasets that are dispersed over multiple databases, websites and literature reports. Mining and integrating these datasets are cumbersome, time-consuming tasks and provide only snapshots of relevant information. To overcome this impediment to effective, efficient study of AR and potential drug targets, we developed the Regulators of Androgen Action Resource (RAAR), a non-redundant, curated and user-friendly searchable web interface. RAAR centralizes information on gene function, clinical relevance, and resources for 55 genes that encode proteins involved in biosynthesis, metabolism and transport of androgens and for 274 AR-associated coregulator genes. Data in RAAR are organized in two levels: (i) Information pertaining to production of androgens is contained in a ‘pre-receptor level’ database, and coregulator gene information is provided in a ‘post-receptor level’ database, and (ii) an ‘other resources’ database contains links to additional databases that are complementary to and useful to pursue further the information provided in RAAR. For each of its 329 entries, RAAR provides access to more than 20 well-curated publicly available databases, and thus, access to thousands of data points. Hyperlinks provide direct access to gene-specific entries in the respective database(s). RAAR is a novel, freely available resource that provides fast, reliable and easy access to integrated information that is needed to develop alternative CaP therapies. Database URL: http://www.lerner.ccf.org/cancerbio/heemers/RAAR/search/

Background: Next-generation sequencing is revealing genomic heterogeneity in localized prostate cancer (CaP). Incomplete sampling of CaP multiclonality has limited the implications for molecular subtyping, stratification, and systemic treatment.Objective: To determine the impact of genomic and transcriptomic diversity within and among intraprostatic CaP foci on CaP molecular taxonomy, predictors of progression, and actionable therapeutic targets.Design, setting, and participants: Four consecutive patients with clinically localized National Comprehensive Cancer Network intermediate-or high-risk CaP who did not receive neoadjuvant therapy underwent radical prostatectomy at Roswell Park Cancer Institute in June-July 2014. Presurgical information on CaP content and a customized tissue procurement procedure were used to isolate nonmicroscopic and noncontiguous CaP foci in radical prostatectomy specimens. Three cores were obtained from the index lesion and one core from smaller lesions. RNA and DNA were extracted simultaneously from 26 cores with >= 90% CaP content and analyzed using whole-exome sequencing, single-nucleotide polymorphism arrays, and RNA sequencing.Outcome measurements and statistical analysis: Somatic mutations, copy number alternations, gene expression, gene fusions, and phylogeny were defined. The impact of genomic alterations on CaP molecular classification, gene sets measured in Oncotype DX, Prolaris, and Decipher assays, and androgen receptor activity among CaP cores was determined.Results and limitations: There was considerable variability in genomic alterations among CaP cores, and between RNA-and DNA-based platforms. Heterogeneity was found in molecular grouping of individual CaP foci and the activity of gene sets underlying the assays for risk stratification and androgen receptor activity, and was validated in independent genomic data sets. Determination of the implications for clinical decision-making requires follow-up studies.Conclusions: Genomic make-up varies widely among CaP foci, so care should be taken when making treatment decisions based on a single biopsy or index lesions.Patient summary: We examined the molecular composition of individual cancers in a patient's prostate. We found a lot of genetic diversity among these cancers, and concluded that information from a single cancer biopsy is not sufficient to guide treatment decisions. (C) 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Abstract The ∼30,000 prostate cancer (CaP) deaths in the United States annually are due to failure of androgen deprivation therapy (ADT). ADT prevents ligand-activation of androgen receptor (AR), which is the main target for treatment of non-organ-confined CaP. ADT fails while CaP remains dependent on AR. Recent NextGen sequencing approaches on ADT-recurrent CaP specimens have confirmed AR amplification, and emergence of gain-of-function AR mutations and forms of AR that do not require ligand-activation. The same sequencing studies also identified a striking enrichment in gain-of-function p53 mutations during ADT. AR and p53 have been recognized as the 2 major genomic drivers of lethal CaP progression, do not interact directly and are not targeted efficiently for therapy in ADT-recurrent CaP. Recently, using a customized gene expression oligoarray, we defined the contribution of 18 clinically relevant coregulators to androgen regulation of 452 AR target genes. Coregulator contribution to AR action was highly gene-specific and context-dependent. Selectivity in dependence of AR target genes on individual coregulators was reflected in the composition of associated AR binding sites, as well as in CaP cell biology and clinical CaP progression that was controlled by these AR target genes. The existence of diverse AR-coregulator-transcription factor (TF) transcriptional codes was evidenced by WDR77-dependent interaction between AR and p53. In this novel AR-, p53- and WDR77-dependent transcriptional mechanism, the coregulator WDR77 physically and functionally bridged the action of AR and p53. WDR77 controlled selectively androgen regulation of 96 AR target genes that are associated with aggressive CaP and that mediate cell death, DNA replication, recombination and repair. Using Cistrome project tools, binding motifs for p53 were found close to AR binding sites in WDR77-dependent AR target genes, and ChIP validated androgen-dependent recruitment of p53 and WDR77 to those genomic regions. Genome-wide gene expression profiling confirmed significant overlap (n = 262) in p53- and WDR77-dependent androgen-responsive genes in CaP cells. Co-immunoprecipitation and mammalian-2-hybrid assays demonstrated p53 and WDR77 interaction, and delineated the p53-WDR77 interacting domains. In conclusion, WDR77-dependent interaction between AR and p53 provides the rationale for a novel CaP treatment strategy, namely disruption of functional interaction(s) between AR and TFs, such as p53, that control the CaP biology responsible for lethal disease progression. Citation Format: Sangeeta Kumari, Simon Schlanger, Dan Wang, Song Liu, Hannelore Heemers. Isolation of WDR77-mediated interaction between androgen receptor and p53 uncovers novel treatment strategy for prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 865.

The ubiquitin ligase CHIP plays an important role in cytosolic protein quality control by ubiquitinating proteins chaperoned by Hsp70/Hsc70 and Hsp90, thereby targeting such substrate proteins for degradation. We present a 2.91 angstrom resolution structure of the tetratricopeptide repeat (TPR) domain of CHIP in complex with the a-helical lid subdomain and unstructured tail of Hsc70. Surprisingly, the CHIP-TPR interacts with determinants within both the Hsc70-lid subdomain and the C-terminal PTIEEVD motif of the tail, exhibiting an atypical mode of interaction between chaperones and TPR domains. We demonstrate that the interaction between CHIP and the Hsc70-lid subdomain is required for proper ubiquitination of Hsp70/Hsc70 or Hsp70/Hsc70-bound substrate proteins. Posttranslational modifications of the Hsc70 lid and tail disrupt key contacts with the CHIP-TPR and may regulate CHIP-mediated ubiquitination. Our study shows how CHIP docks onto Hsp70/Hsc70 and defines a bipartite mode of interaction between TPR domains and their binding partners.