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Measurable residual disease (MRD) is a powerful prognostic factor in acute myeloid leukemia (AML). However, pre-treatment molecular predictors of immunophenotypic MRD clearance remain unclear. We analyzed a dataset of 211 patients with pre-treatment next-generation sequencing who received induction chemotherapy and had MRD assessed by serial immunophenotypic monitoring after induction, subsequent therapy, and allogeneic stem cell transplant (allo-SCT). Induction chemotherapy led to MRD- remission, MRD+ remission, and persistent disease in 35%, 27%, and 38% of patients, respectively. With subsequent therapy, 34% of patients with MRD+ and 26% of patients with persistent disease converted to MRD-. Mutations in CEBPA, NRAS, KRAS, and NPM1 predicted high rates of MRD- remission, while mutations in TP53, SF3B1, ASXL1, and RUNX1 and karyotypic abnormalities including inv (3), monosomy 5 or 7 predicted low rates of MRD- remission. Patients with fewer individual clones were more likely to achieve MRD- remission. Among 132 patients who underwent allo-SCT, outcomes were favorable whether patients achieved early MRD- after induction or later MRD- after subsequent therapy prior to allo-SCT. As MRD conversion with chemotherapy prior to allo-SCT is rarely achieved in patients with specific baseline mutational patterns and high clone numbers, upfront inclusion of these patients into clinical trials should be considered.

Acute myeloid leukaemia (AML) represents a set of heterogeneous myeloid malignancies, and hallmarks include mutations in epigenetic modifiers, transcription factors and kinasessup1-5/sup. The extent to which mutations in AML drive alterations in chromatin 3D structure and contribute to myeloid transformation is unclear. Here we use Hi-C and whole-genome sequencing to analyse 25 samples from patients with AML and 7 samples from healthy donors. Recurrent and subtype-specific alterations in A/B compartments, topologically associating domains and chromatin loops were identified. RNA sequencing, ATAC with sequencing and CUTamp;Tag for CTCF, H3K27ac and H3K27me3 in the same AML samples also revealed extensive and recurrent AML-specific promoter-enhancer and promoter-silencer loops. We validated the role of repressive loops on their target genes by CRISPR deletion and interference. Structural variation-induced enhancer-hijacking and silencer-hijacking events were further identified in AML samples. Hijacked enhancers play a part in AML cell growth, as demonstrated by CRISPR screening, whereas hijacked silencers have a downregulating role, as evidenced by CRISPR-interference-mediated de-repression. Finally, whole-genome bisulfite sequencing of 20 AML and normal samples revealed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Treatment of AML cells with a DNA hypomethylating agent and triple knockdown of DNMT1, DNMT3A and DNMT3B enabled the manipulation of DNA methylation to revert 3D genome organization and gene expression. Overall, this study provides a resource for leukaemia studies and highlights the role of repressive loops and hijacked cis elements in human diseases.

In adult acute myeloid leukemia (AML), the acquisition of driver somatic mutations may be preceded by a benign state termed clonal hematopoiesis (CH). To develop therapeutic strategies to prevent leukemia development from CH, it is important to understand the mechanisms by which CH-driving and AML-driving mutations cooperate. Here, we use mice with inducible mutant alleles common in human CH (DNMT3AR882; mouse Dnmt3aR878H) and AML (NPM1c; mouse Npm1cA). We find that Dnmt3aR878H/+ hematopoietic stem cells (HSCs), but not multipotent progenitor cell (MPP) subsets, have reduced cytokine expression and proinflammatory transcriptional signatures and a functional competitive advantage over their wild-type counterparts. Dnmt3aR878H/+ HSCs are the most potent cell type transformed by Npm1cA, generating myeloid malignancies in which few additional cooperating somatic mutation events were detected. At a molecular level, Npm1cA, in cooperation with Dnmt3aR878H, acutely increased the accessibility of a distinct set of promoters in HSCs compared with MPP cells. These promoters were enriched for cell cycling, PI3K/AKT/mTOR signaling, stem cell signatures, and targets of transcription factors, including NFAT and the chromatin binding factor HMGB1, which have been implicated in human AML. These results demonstrate cooperativity between preexisting Dnmt3aR878H and Npm1cA at the chromatin level, where specific loci altered in accessibility by Npm1cA are dependent on cell context as well as Dnmt3a mutation status. These findings have implications for biological understanding and therapeutic intervention in the transformation from CH to AML.

Purpose: To study the biology and identify markers of severe cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS) in children after chimeric antigen receptor T-cell (CAR T) treatment. Experimental Design: We used comprehensive proteomic profiling to measure over 1,400 serum proteins at multiple serial timepoints in a cohort of patients with B-cell acute lymphoblastic leukemia treated with the CD19-targeted CAR T CTL019 on two clinical trials. Results: We identified fms-like tyrosine kinase 3 (FLT3) and mast cell immunoglobulin-like receptor 1 (MILR1) as preinfusion predictive biomarkers of severe CRS. We demonstrated that CRS is an IFNγ-driven process with a protein signature overlapping with hemophagocytic lymphohistiocytosis (HLH). We identified IL18 as a potentially targetable cytokine associated with the development of ICANS. Conclusions: We identified preinfusion biomarkers that can be used to predict severe CRS with a sensitivity, specificity, and accuracy superior to the current gold standard of disease burden. We demonstrated the fundamental role of the IFNγ pathway in driving CRS, suggesting CRS and carHLH are overlapping rather than distinct phenomena, an observation with important treatment implications. We identified IL18 as a possible targetable cytokine in ICANS, providing rationale for IL18 blocking therapies to be translated into clinical trials in ICANS.

We hypothesized that combining adoptively transferred autologous T cells with a cancer vaccine strategy would enhance therapeutic efficacy by adding antimyeloma idiotype (Id)–keyhole limpet hemocyanin (KLH) vaccine to vaccine-specific costimulated T cells. In this randomized phase 2 trial, patients received either control (KLH only) or Id-KLH vaccine, autologous transplantation, vaccine-specific costimulated T cells expanded ex vivo, and 2 booster doses of assigned vaccine. In 36 patients (KLH, n = 20; Id-KLH, n = 16), no dose-limiting toxicity was seen. At last evaluation, 6 (30%) and 8 patients (50%) had achieved complete remission in KLH-only and Id-KLH arms, respectively (P = .22), and no difference in 3-year progression-free survival was observed (59% and 56%, respectively; P = .32). In a 594 Nanostring nCounter gene panel analyzed for immune reconstitution (IR), compared with patients receiving KLH only, there was a greater change in IR genes in T cells in those receiving Id-KLH relative to baseline. Specifically, upregulation of genes associated with activation, effector function induction, and memory CD8+ T-cell generation after Id-KLH but not after KLH control vaccination was observed. Similarly, in responding patients across both arms, upregulation of genes associated with T-cell activation was seen. At baseline, all patients had greater expression of CD8+ T-cell exhaustion markers. These changes were associated with functional Id-specific immune responses in a subset of patients receiving Id-KLH. In conclusion, in this combination immunotherapy approach, we observed significantly more robust IR in CD4+ and CD8+ T cells in the Id-KLH arm, supporting further investigation of vaccine and adoptive immunotherapy strategies. This trial was registered at www.clinicaltrials.gov as #NCT01426828.

Introduction Subcutaneous injection of testosterone has been investigated as a method of testosterone replacement therapy (TRT) in hypogonadal men. We have been investigating the use of a non-proprietary subcutaneous (SubQ) injection of testosterone cypionate using a ½ inch 27g needle for replacement to treat hypogonadism. This is an update on our work which includes a larger sample size and longer follow up. Objective To assess the use of non-proprietary SubQ TRT as treatment in hypogonadal men and its effect on HCT, PSA, and patient satisfaction. Methods This study is a retrospective chart review of patients receiving SubQ TRT with a mean follow up length of 9.8 months (range 0-24 months). Patient data was collected prior to initiation of therapy and at subsequent visits. Testosterone, HCT, and PSA were determined through regular blood draws and a patient satisfaction survey was performed over the phone. Univariate analysis and paired t-tests were used to analyze study variables. Results 41 total patients received SubQ TRT. Mean changes in total testosterone level (increased 281.3 + 81.1, p-value=0.0008), hematocrit (increased 1.5 + 1.0), PSA (increased 0.4 + 0.4), SHIM score (increased 3.8 + 1.4, p-value=0.0088) and AUA symptom score (decreased 0.9 + 1.6) were calculated at the patients’ initial visit and most recent follow up while still on SubQ TRT (Table 1). A total of 30 patients had been on various combinations of TRT before switching to SubQ including IM (13), CC (13) Testopel (8), HCG (2) SubQ (2), T gel (1) and Anastrozole (1). Data on the most frequent TRTs is demonstrated in Table 2. Patient satisfaction surveys were completed by 32 patients (78%), with 26 (81.2%) patients reporting overall satisfaction with SubQ TRT versus 45% (9/20) with past forms of TRT. 93.7% (15/16) reported SubQ TRT was both easier and less painful, and 73.6% (14/19) preferred SubQ injections over their previous form of TRT. Reasons for stopping SubQ TRT included unrelated stroke (1), prostate cancer (1), development of scar tissue (1) or bruising (1), ineffective therapy (1), difficulty drawing up the syringe (1), cost (1), and trouble obtaining medication from the pharmacy (1). Conclusions The use of non-proprietary testosterone cypionate SubQ as treatment for hypogonadism results in an elevation in serum total testosterone and improvement on both SHIM and AUA symptom scores at follow up. PSA and HCT rose over the span of treatment, but the rises were not statistically significant, and no patient developed erythrocytosis. Our data demonstrate SubQ TRT is easier and less painful to use and scores higher on overall patient satisfaction compared to other forms of TRT. Disclosure No

Introduction Penuma is a novel soft silicone penile sleeve implant that is FDA cleared for cosmetic augmentation of the penis, including enhancing the girth and length of the flaccid penis, and the correction of cosmetic deformities of the penis, such as retractile penis and mid-shaft indentations. Since its FDA clearance, approximately 5,000 Penuma devices have been implanted. The increasing popularity of the implant is accompanied by concerns over patient safety. Possible risks and complications include infection or erosion requiring removal of the implant, hematoma, seroma, prosthesis displacement, and persistent flaring of the Penuma proximal to the coronal sulcus necessitating revision. Over time, the surgical method to implant Penuma has evolved, most notably with the use of a scrotal lateral incision in lieu of an infrapubic incision. Objective Our objective is to provide recent multi-institutional data on safety and efficacy outcomes in patients undergoing Penuma penile implant surgery across multiple high volume implant surgeons at tertiary care centers. Methods We performed a retrospective analysis of men undergoing Penuma implants between November 2020 and March 2022 with three surgeons, each working at a separate large academic hospital. Adverse events including infection and unsatisfactory cosmetic outcomes requiring revision as determined by physician follow up and patient questionnaires were recorded. Outcomes include measurements of incidents of peri and post-operative adverse events and penile length and girth pre- and post-operatively (Figure 1). Results 169 male patients underwent Penuma implant surgery. Mean age was 42.3 +/− 9.8 years. Mean BMI was 28.7 +/− 4.1. Our patient population was very healthy (3% tobacco smokers, 2% diabetes, 1% cardiovascular disease). Pre-operative mean flaccid length was 7.6 +/− 1.9 cm. Post-operative mean length was 11.2 +/− 1.9 cm. Patients added an average of 3.8 +/− 1.9 cm to their penile length, a 50% increase (p < 0.001). Pre-operative mean flaccid girth was 9.1 +/− 1.4 cm. Post-operative mean girth was 12.5 +/− 1.2 cm. Patients added an average of 3.4 +/− 1.1 cm to their penile girth, a 37% increase (p < 0.001). Complications rates were: infection 0.6%, seroma 1.2%, and erosion 3.6% (Figure 2). Complication rates were found to be lower than those reported in a previously published, IRB-approved retrospective review of Penuma patient outcomes. Ten patients (5.9%) reported unsatisfactory cosmetic results - all had persistent flaring of the Penuma at the coronal sulcus; all but one opted for revision surgery (Figure 3). Based on patient questionnaires, all revisions had satisfactory cosmetic outcomes. One revision was completed under local anesthesia at the time of scrotoplasty. Conclusions The Penuma implant can be used to safely enhance flaccid penile length and girth in patients with retractile penis or other cosmetic deformities and forms of cosmetic augmentation. Should complications occur, they are mainly cosmetic. Their correction is completed easily and with low risk. Disclosure No

Introduction Peyronie’s disease (PD) is a progressive wound-healing disorder resulting in plaque formation within the tunica albuginea. PD continues to be poorly understood and an area of investigation, with increased awareness by patients and clinicians. Most commonly, PD patients will have a singular plaque and uniplanar curvature in the dorsal or lateral direction. However, previous studies have categorized less common deformities as “atypical”, which includes ventral curvature, multiplanar curvatures, unilateral indentations, hourglass deformity, and severe loss of penile length. Objective We aim to characterize the presenting features of PD within a large cohort of patients presenting to a tertiary referral center. Furthermore, we seek to elucidate the pre-operative factors that correlate with subsequent surgical intervention. Methods 1483 consecutive patients presenting with PD from 2016 to 2021 were retrospectively identified. Patients were excluded if they only had virtual visits, refused penile duplex evaluation, or had PD surgical correction prior to presentation. Baseline demographics, prior treatments, physical exam findings, penile duplex assessment (PDDU), subsequent interventions, and clinical outcomes were documented. Univariate and multivariate analyses were performed to assess pre-operative predictors of subsequent surgical intervention. Results In total, 1263 patients met inclusion criteria. Mean age at presentation was 55.4 ± 11.1 years with a mean duration of PD at presentation of 33.2 ± 42.5 months. Mean primary curvature was 49.8 ± 20.8°. Primary ventral curvature was present in 11.4% and 36.5% of patients had a multiplanar curvature. Mean composite curvature was 60.4 ± 28.2°. Subjective penile shortening was reported by 64.8% of patients with a mean reported loss of 3.8 ± 2.1 cm. During PDDU, indentation/narrowing deformities was appreciated in 73.7%, hourglass deformity in 10.1%, and hinge effect in 33.0% of patients. Calcification was seen in 30.1% of patients. Operative intervention occurred in 35.3% of patients. Hypogonadism (1.56 OR, p=0.04), severity of primary curvature (1.03 OR, p Conclusions Peyronie’s disease remains an incompletely understood disease and presenting features may be changing as a result of more sophisticated evaluation and physician experience. Ventral curvature, multiplanar curvatures, indentation deformity, hourglass deformity, and severe loss of penile length have been described as “atypical”, but these changes are not rare in our cohort of referred patients. Patients with worse erectile function and more severe PD characteristics were predictors of surgical intervention. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Boston Scientific.

Introduction Peyronie’s Disease (PD) is a debilitating condition characterized by progressive scarring of the tunica albuginea. The resulting deformities are often emotionally devastating to patients, and include erectile dysfunction, painful erections, penile curvature, indentation, hourglass, and hinge deformities. According to previously published surgical algorithm for PD in 1997, partial plaque excision and grafting (PEG) is recommended for patients with >60 degree curvature +/− hinge and strong pre-operative erections while placement of an inflatable penile prosthesis (IPP) is recommended when rigidity is sub-optimal.* Nevertheless, many patients counseled to undergo an IPP refuse and insist on proceeding with PEG due to personal preference. We hypothesize that men counseled to undergo IPP but choose to undergo PEG will report lower satisfaction rates and reduced ability to engage in sexual intercourse post-operatively. Objective Our objective was to evaluate and compare long-term patient-reported outcomes in men who underwent PEG at a single institution between 2007 and 2021. Methods A retrospective chart review was conducted from 2007 to 2021 on PEG surgeries performed at a single, tertiary-care institution in patients ≥18 years old who had >3 months of follow up. Results 251 patients underwent PEG with a mean follow up was 35.5 months. 54 patients (22%) were initially advised to undergo IPP but elected for PEG. Of these 54 patients, 17 (32%) had pre-operative 9-10 subjective erectile rigidity, relative to 110 of 197 (56%) patients who were initially counseled to undergo PEG (p=0.002). On duplex penile ultrasound, patients counseled to undergo IPP had a mean objective erectile rigidity of 7.8, compared to a mean objective rigidity of 8.6 in patients initially counseled to undergo PEG (p 15 units for pre-operative duplex ultrasound evaluation were less likely to be able to engage in post-operative intercourse following PEG surgery (48.6%) relative to those who needed Conclusions Patients who were initially counseled to undergo IPP due to sub-optimal erectile rigidity but elected for PEG procedure had worse post-operative ability to engage in penetrative intercourse. Significantly more of these patients also eventually underwent IPP. Pre-operative trimix dosing Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Boston Scientific (Consultant)

Introduction Peyronie’s disease (PD) is a disorder marked by the formation of a fibrous plaque in the tunica albuginea often causing a penile deformity. Surgical correction remains the gold standard of treatment for PD. The choice of procedure depends on various factors including erectile function, degree of penile curvature, hinge effect, and patient preference. We have found that men undergoing surgical correction of PD had intraoperative identification of a glans tilt or floppy glans, which was not recognized preoperatively. Objective Our aim was to analyze a large PD database to review the results after a glanspexy was performed in men undergoing partial plaque excision and grafting (PEG), tunica albuginea plication (TAP) or inflatable penile prosthesis (IPP) with straightening maneuvers. Methods A retrospective analysis was performed on all patients who presented with PD and were subsequently treated with a surgical procedure that included a glanspexy from 2016-2021. Demographics, comorbidities, erectile function (ED), PD characteristics, physical exam, penile duplex ultrasound findings, and surgical outcomes were obtained. Patients were tracked longitudinally, and subsequent interventions and outcomes were collected. All glanspexies were performed using the same surgical technique, where the glans was secured over the tips of the corpora cavernosa using several 4-0 Monocryl sutures between the glans and the tunica albuginea contralateral to the tilt through a subcoronal incision. Results A total of 97 glanspexies were performed in conjunction with an operation for PD. IPP with glanspexy (49.5%) was the most common procedure performed followed by PEG with glanspexy (36.1%) and TAP with glanspexy (14.4%). Post-operatively, there were no patient complaints or physician identification of irregularities pertaining to glans position or sensitivity. 6 patients required a reoperation after their initial PD procedure, however, none of the indications were related to the glanspexy portion of the procedure. There was one patient who sustained partial glans necrosis following complex reconstruction including extensive plaque incision with grafting, IPP and glanspexy. Conclusions Glanspexy using 4-0 Monocryl suture on a TF needle appears to be a safe, easy and effective technique for the treatment of a glans tilt or floppy glans identified during the surgical management of PD. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Boston Scientific.

Acute myeloid leukemia (AML) is an aggressive disease with complex and heterogeneous biology. Although several genomic classifications have been proposed, there is a growing interest in going beyond genomics to stratify AML. In this study, we profile the sphingolipid family of bioactive molecules in 213 primary AML samples and 30 common human AML cell lines. Using an integrative approach, we identify two distinct sphingolipid subtypes in AML characterized by a reciprocal abundance of hexosylceramide (Hex) and sphingomyelin (SM) species. The two Hex-SM clusters organize diverse samples more robustly than known AML driver mutations and are coupled to latent transcriptional states. Using transcriptomic data, we develop a machine-learning classifier to infer the Hex-SM status of AML cases in TCGA and BeatAML clinical repositories. The analyses show that the sphingolipid subtype with deficient Hex and abundant SM is enriched for leukemic stemness transcriptional programs and comprises an unappreciated high-risk subgroup with poor clinical outcomes. Our sphingolipid-focused examination of AML identifies patients least likely to benefit from standard of care and raises the possibility that sphingolipidomic interventions could switch the subtype of AML patients who otherwise lack targetable alternatives.Key Points1.Sphingolipidomics separates acute myeloid leukemia (AML) patients and cell lines into two subtypes.2.The subtype with low hexosylceramide and high sphingomyelin defines a new high-risk subtype with poor clinical outcomes.

Supplementary Data from PU.1-Dependent Enhancer Inhibition Separates Tet2-Deficient Hematopoiesis from Malignant Transformation

Supplementary Figure from PU.1-Dependent Enhancer Inhibition Separates Tet2-Deficient Hematopoiesis from Malignant Transformation

Cytosine hypermethylation in and around DNA-binding sites of master transcription factors, including PU.1, occurs in aging hematopoietic stem cells following acquired loss-of-function mutations of DNA methyl-cytosine dioxygenase ten–eleven translocation-2 (TET2), albeit functional relevance has been unclear. We show that Tet2-deficient mouse hematopoietic stem and progenitor cells undergo malignant transformation upon compromised gene regulation through heterozygous deletion of an upstream regulatory region (UREΔ/WT) of the PU.1 gene. Although compatible with multilineage blood formation at young age, Tet2-deficient PU.1 UREΔ/WT mice develop highly penetrant, transplantable acute myeloid leukemia (AML) during aging. Leukemic stem and progenitor cells show hypermethylation at putative PU.1-binding sites, fail to activate myeloid enhancers, and are hallmarked by a signature of genes with impaired expression shared with human AML. Our study demonstrates that Tet2 and PU.1 jointly suppress leukemogenesis and uncovers a methylation-sensitive PU.1-dependent gene network as a unifying molecular vulnerability associated with AML.Significance:We identify moderately impaired PU.1 mRNA expression as a biological modality predisposing Tet2-deficient hematopoietic stem and progenitor cells to malignant transformation. Our study furthermore uncovers a methylation-sensitive PU.1 gene network as a common feature of myeloid leukemia potentially allowing for the identification of patients at risk for malignant transformation.See related commentary by Schleicher and Pietras, p. 378.This article is highlighted in the In This Issue feature, p. 369

supplementary tables and material and methods

Supplementary Methods, Figure Legends

Patient information of all AML subtypes

Events upstream AID-mediated switch lesion are not affected in Nbs1deltaB/deltaB Atm -/- VAV B cells

Flow cytometric analysis of haematopoietic tissues

PDF file 338K, Phospho-flow data

The success of chimeric antigen receptor (CAR)–mediated immunotherapy in acute lymphoblastic leukemia (ALL) highlights the potential of T-cell therapies with directed cytotoxicity against specific tumor antigens. The efficacy of CAR T-cell therapy depends on the engraftment and persistence of T cells following adoptive transfer. Most protocols for T-cell engineering routinely expand T cells ex vivo for 9 to 14 days. Because the potential for engraftment and persistence is related to the state of T-cell differentiation, we hypothesized that reducing the duration of ex vivo culture would limit differentiation and enhance the efficacy of CAR T-cell therapy. We demonstrated that T cells with a CAR-targeting CD19 (CART19) exhibited less differentiation and enhanced effector function in vitro when harvested from cultures at earlier (day 3 or 5) compared with later (day 9) timepoints. We then compared the therapeutic potential of early versus late harvested CART19 in a murine xenograft model of ALL and showed that the antileukemic activity inversely correlated with ex vivo culture time: day 3 harvested cells showed robust tumor control despite using a 6-fold lower dose of CART19, whereas day 9 cells failed to control leukemia at limited cell doses. We also demonstrated the feasibility of an abbreviated culture in a large-scale current good manufacturing practice–compliant process. Limiting the interval between T-cell isolation and CAR treatment is critical for patients with rapidly progressing disease. Generating CAR T cells in less time also improves potency, which is central to the effectiveness of these therapies. Cancer Immunol Res; 6(9); 1100–9. ©2018 AACR.

Next-generation sequencing and droplet digital PCR

Fig. S1. CARTmeso cell manufacturing and treatment schedules. Fig. S2. Detection of human anti-chimeric antibody responses in patients post-CARTmeso cell infusion. Fig. S3. Serum cytokines and chemokines in PDA patient 21211-101. Fig. S4. CARTmeso cytolytic activity against allogeneic and autologous pancreatic cancer cell lines Table S1. Patient demographics. Table S2. Treatment-related adverse events. Table S3. Protoarray analysis of serum samples from mesothelioma patient 17510-105 Table S4. Protoarray analysis of serum samples from pancreatic cancer patient 21211-101

Epigenetic allele diversity is linked to inferior prognosis in acute myeloid leukemia (AML). However, the source of epiallele heterogeneity in AML is unknown. Herein we analyzed epiallele diversity in a genetically and clinically annotated AML cohort. Notably, AML driver mutations linked to transcription factors and favorable outcome are associated with epigenetic destabilization in a defined set of susceptible loci. In contrast, AML subtypes linked to inferior prognosis manifest greater abundance and highly stochastic epiallele patterning. We report an epiallele outcome classifier supporting the link between epigenetic diversity and treatment failure. Mouse models with TET2 or IDH2 mutations show that epiallele diversity is especially strongly induced by IDH mutations, precedes transformation to AML, and is enhanced by cooperation between somatic mutations. Furthermore, epiallele complexity was partially reversed by epigenetic therapies in AML driven by TET2/IDH2, suggesting that epigenetic therapy might function in part by reducing population complexity and fitness of AMLs.Significance:We show for the first time that epigenetic clonality is directly linked to specific mutations and that epigenetic allele diversity precedes and potentially contributes to malignant transformation. Furthermore, epigenetic clonality is reversible with epigenetic therapy agents.This article is highlighted in the In This Issue feature, p. 1775

XLSX file - 24K, PCR Primer sequences.

PDF file 103K, Supplementary tables and supplementary figure legends

shRNA screen hits

Supplementary Figure from LKB1/STK11 Is a Tumor Suppressor in the Progression of Myeloproliferative Neoplasms

PDF file - 113K, Bcl2 protein expression in wild-type and Tyk2 knockout mice.

PDF file - 841K, Supplementary Figures S1-S6; legends for Supplementary Tables S1-S3; Supplementary Methods describing RNA interference, generation of Ba/F3 cell lines, site-directed mutagenesis, transient transfection, inhibition of cellular ATP production, proliferation assays, enzymatic assays with JAK2 JH1, purification of tyrosine phosporylated peptides and mass-spectrometry; Supplementary References.

Supplementary Figures S1-S6

PDF file - 74K, Knockdown efficiency and BCL2 expression in JURKAT cells.