Oncogenic Fusion Protein Research Articles

EWSR1 maintains centromere identity

The centromere is essential for ensuring high-fidelity transmission of chromosomes. CENP-A, the centromeric histone H3 variant, is thought to be the epigenetic mark of centromere identity. CENP-A deposition at the centromere is crucial for proper centromere function and inheritance. Despite its importance, the precise mechanism responsible for maintenance of centromere position remains obscure. Here, we report a mechanism to maintain centromere identity. We demonstrate that CENP-A interacts with EWSR1 (Ewing sarcoma breakpoint region 1) and EWSR1-FLI1 (the oncogenic fusion protein in Ewing sarcoma). EWSR1 is required for maintaining CENP-A at the centromere in interphase cells. EWSR1 and EWSR1-FLI1 bind CENP-A through the SYGQ2 region within the prion-like domain, important for phase separation. EWSR1 binds to R-loops through its RNA-recognition motif invitro. Both the domain and motif are required for maintaining CENP-A at the centromere. Therefore, we conclude that EWSR1 guards CENP-A in centromeric chromatins by binding to centromeric RNA.

Development of the nonreceptor tyrosine kinase FER-targeting PROTACs as a potential strategy for antagonizing ovarian cancer cell motility and invasiveness

Aberrant overexpression of nonreceptor tyrosine kinase FER (Fps/Fes Related) has been reported in various ovarian carcinoma-derived tumor cells and is a poor prognosis factor for patient survival. It plays an essential role in tumor cell migration and invasion, acting concurrently in both kinase-dependent and -independent manners, which is not easily suppressed by conventional enzymatic inhibitors. Nevertheless, the PROteolysis-TArgeting Chimera (PROTAC) technology offers superior efficacy over traditional activity-based inhibitors by simultaneously targeting enzymatic and scaffold functions. Hence in this study, we report the development of two PROTAC compounds that promote robust FER degradation in a cereblon-dependent manner. Both PROTAC degraders outperform a Food and Drug Administration-approved drug, brigatinib, in ovarian cancer cell motility suppression. Importantly, these PROTAC compounds also degrade multiple oncogenic FER fusion proteins identified in human tumor samples. These results lay an experimental foundation to apply the PROTAC strategy to antagonize cell motility and invasiveness in ovarian and other types of cancers with aberrant expression of FER kinase and highlight PROTACs as a superior strategy for targeting proteins with multiple tumor-promoting functions.

Palbociclib in Acute Leukemias With KMT2A-rearrangement: Results of AMLSG 23-14 Trial.

Palbociclib in Acute Leukemias With KMT2A-rearrangement: Results of AMLSG 23-14 Trial.

Cell state-dependent chromatin targeting in NUT carcinoma.

Aberrant transcriptional programming and chromatin dysregulation are common to most cancers. Whether by deranged cell signaling or environmental insult, the resulting oncogenic phenotype is typically manifested in transcriptional changes characteristic of undifferentiated cell growth. Here we analyze targeting of an oncogenic fusion protein, BRD4-NUT, composed of 2 normally independent chromatin regulators. The fusion causes the formation of large hyperacetylated genomic regions or megadomains, mis-regulation of c-MYC, and an aggressive carcinoma of squamous cell origin. Our previous work revealed largely distinct megadomain locations in different NUT carcinoma patient cell lines. To assess whether this was due to variations in individual genome sequences or epigenetic cell state, we expressed BRD4-NUT in a human stem cell model and found that megadomains formed in dissimilar patterns when comparing cells in the pluripotent state with the same cell line following induction along a mesodermal lineage. Thus, our work implicates initial cell state as the critical factor in the locations of BRD4-NUT megadomains. These results, together with our analysis of c-MYC protein-protein interactions in a patient cell line, are consistent with a cascade of chromatin misregulation underlying NUT carcinoma.

Cell cycle arrest and apoptosis are early events in radiosensitization of EWS::FLI1+ Ewing sarcoma cells by Mithramycin A

Purpose The oncogenic fusion protein EWS::FLI1 is an attractive therapeutic target in Ewing sarcoma (ES). Mithramycin A (MithA) is a potent and specific inhibitor of EWS::FLI1 that can selectively radiosensitize ES cells through transcriptional inhibition of DNA double-strand break (DSB) repair. Here, we evaluate temporal changes in cell cycle progression and apoptosis in ES cells treated with MithA and/or ionizing radiation (RTx), testing the hypothesis that combining MithA with ionizing radiation would synergistically impair cell cycle progression and enhance apoptotic elimination to a greater extent than either agent alone. Materials and Methods Four EWS::FLI1 + ES cell lines TC-71, RD-ES, SK-ES-1, and A673, and one EWS::ERG cell line (CHLA-25) were exposed to 10nM MithA or vehicle and followed 24 h later by exposure to 2 Gy x-radiation or sham irradiation. Reactive oxygen species (ROS) activity was evaluated by cytometric assay, and assay of antioxidant gene expression by RT-qPCR. Cell cycle changes were evaluated by flow cytometry of nuclei stained with propidium iodide. Apoptosis was assessed by cytometric assessment of Caspase-3/7 activity and by immunoblotting of PARP-1 cleavage. Radiosensitization was evaluated by clonogenic survival assay. Proliferation (EdU) and apoptosis (TUNEL) were evaluated in SK-ES-1 xenograft tumors following pretreatment with 1 mg/kg MithA, followed 24 h later by a single 4 Gy fraction of x-radiation. Results MithA-treated cells showed reduced levels of ROS, and were associated with increased expression of antioxidant genes SOD1, SOD2, and CAT. It nonetheless induced persistent G0/G1 arrest and a progressive increase of the sub-G1 fraction, suggesting apoptotic degeneration. In vitro assays of Caspase-3/7 activity and immunoblotting of Caspase-3/7 dependent cleavage of PARP-1 indicated that apoptosis began as early as 24 h after MithA exposure, reducing clonogenic survival. Tumors from xenograft mice treated with either radiation alone, or in combination with MithA showed a significant reduction of tumor cell proliferation, while apoptosis was significantly increased in the group receiving the combination of MithA and RTx. Conclusions Taken together, our data show that the anti-proliferative and cytotoxic effects of MithA are the prominent components of radiosensitization of EWS::FLI1+ ES, rather than the result of acutely enhanced ROS levels.

Abstract LB034: EWSR1, Ewing sarcoma breakpoint region 1, maintains centromere identity by binding to centromeric R-loops

Abstract The centromere DNA-kinetochore complex is the specialized chromatin structure that mediates chromosome attachment to microtubules and is required for high-fidelity chromosome transmission. CENP-A is the centromere-specific histone H3 variant that epigenetically determines centromere position on the chromosome to ensure kinetochore assembly. Despite its importance, the precise mechanism of maintenance of CENP-A, i.e., the maintenance of centromere identity, remains obscure. In this study, we found that EWSR1 (Ewing Sarcoma Breakpoint Region 1) is required for CENP-A deposition at centromeres by binding to centromeric RNA. We determined that the CENP-A binding domain of EWSR1 is the SYGQ2 region within the prion-like domain, which plays an important role in phase separation for chromatin remodeling. Our data suggest that EWSR1 guards CENP-A in centromeric chromatins by binding to centromeric RNA. This is a novel mechanism to maintain the position of the centromere on the chromosome i.e., centromere identify. As EWSR1-FLI1 is the oncogenic fusion protein in Ewing sarcoma and as it inhibits EWSR1 function as a dominant negative mutant, it contributes to chromosome instability in Ewing sarcoma, which may lead to therapy resistance and metastasis. Citation Format: Risa Kitagawa, Argentina Becker, Peter Houghton, Katsumi Kitagawa. EWSR1, Ewing sarcoma breakpoint region 1, maintains centromere identity by binding to centromeric R-loops [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB034.

RNA fusion transcript panel identifies diverse repertoire of fusions in adult glioma patients with therapeutic implications.

Recurrent gliomas are therapeutically challenging diseases with few treatment options available. One area of potential therapeutic vulnerability is the presence of targetable oncogenic fusion proteins. To better understand the clinical benefit of routinely testing for fusion proteins in adult glioma patients, we performed a retrospective review of 647 adult patients with glioma who underwent surgical resection at our center between August 2017 and May 2021 and whose tumors were analyzed with an in-house fusion transcript panel. Fifty-two patients (8%) were found to harbor a potentially targetable fusion with 11 (21%) of these patients receiving treatment with a fusion-targeted inhibitor. The targetable genes found to be involved in a fusion included FGFR3, MET, EGFR, NTRK1, NTRK2, BRAF, ROS1, and PIK3CA. This analysis demonstrates that routine clinical testing for gene fusions identifies a diverse repertoire of potential therapeutic targets in adult patients with glioma and can offer rational therapeutic options for patients with recurrent disease.

De Novo Linear Phosphorylation Site Motifs for BCR-ABL Kinase Revealed by Phospho-Proteomics in Yeast.

BCR-ABL is the oncogenic fusion product of tyrosine kinase ABL1 and a highly frequent driver of acute lymphocytic leukemia (ALL) and chronic myeloid leukemia (CML). The kinase activity of BCR-ABL is strongly elevated; however, changes of substrate specificity in comparison to wild-type ABL1 kinase are less well characterized. Here, we heterologously expressed full-length BCR-ABL kinases in yeast. We exploited the proteome of living yeast as an in vivo phospho-tyrosine substrate for assaying human kinase specificity. Phospho-proteomic analysis of ABL1 and BCR-ABL isoforms p190 and p210 yielded a high-confidence data set of 1127 phospho-tyrosine sites on 821 yeast proteins. We used this data set to generate linear phosphorylation site motifs for ABL1 and the oncogenic ABL1 fusion proteins. The oncogenic kinases yielded a substantially different linear motif when compared to ABL1. Kinase set enrichment analysis with human pY-sites that have high linear motif scores well-recalled BCR-ABL driven cancer cell lines from human phospho-proteome data sets.

Melatonin promotes differentiation and apoptosis of AML1-ETO-positive cells.

Acute Myeloid Leukemia 1-Eight-Twenty-One (AML1-ETO) is an oncogenic fusion protein that causes acute myeloid leukemia. We examined the effects of melatonin on AML1-ETO by investigating cell differentiation, apoptosis, and degradation in leukemia cell lines. We evaluated Kasumi-1, U937T, and primary acute myeloid leukemia (AML1-ETO-positive) cell proliferation by Cell Counting Kit-8 assay. Flow cytometry and western blotting were used to evaluate CD11b/CD14 levels (differentiation biomarkers) and the AML1-ETO protein degradation pathway, respectively. CM-Dil-labeled Kasumi-1 cells were also injected into zebrafish embryos to determine the effects of melatonin on vascular proliferation and development and to evaluate the combined effects of melatonin and common chemotherapeutic agents. AML1-ETO-positive acute myeloid leukemia cells were more sensitive to melatonin than AML1-ETO-negative cells. Melatonin increased apoptosis and CD11b/CD14 expression in AML1-ETO-positive cells and decreased the nuclear/cytoplasmic ratio, together suggesting that melatonin induced cell differentiation. Mechanistically, melatonin degraded AML1-ETO by activating the caspase-3 pathway and regulating the mRNA levels of AML1-ETO downstream genes. Melatonin reduced the number of neovessels in Kasumi-1-injected zebrafish, suggesting that melatonin inhibits cell proliferation in vivo. Finally, combining drugs with melatonin inhibited cell viability. Melatonin is a potential compound for the treatment of AML1-ETO-positive acute myeloid leukemia.

Selpercatinib: First approved selective RET inhibitor

Selpercatinib is a small molecule that binds at the RET kinase active site. It inhibits activity of constitutively dimerized RET fusion proteins and activated point mutants, thereby blocking downstream signals for proliferation and survival. It is the first selective RET inhibitor to be FDA approved for tumor agnostic targeting of oncogenic RET fusion proteins. To view this Bench to Bedside, open or download the PDF.

Highly connected 3D chromatin networks established by an oncogenic fusion protein shape tumor cell identity

Cell fate transitions observed in embryonic development involve changes in three-dimensional genomic organization that provide proper lineage specification. Whether similar events occur within tumor cells and contribute to cancer evolution remains largely unexplored. We modeled this process in the pediatric cancer Ewing sarcoma and investigated high-resolution looping and large-scale nuclear conformation changes associated with the oncogenic fusion protein EWS-FLI1. We show that chromatin interactions in tumor cells are dominated by highly connected looping hubs centered on EWS-FLI1 binding sites, which directly control the activity of linked enhancers and promoters to establish oncogenic expression programs. Conversely, EWS-FLI1 depletion led to the disassembly of these looping networks and a widespread nuclear reorganization through the establishment of new looping patterns and large-scale compartment configuration matching those observed in mesenchymal stem cells, a candidate Ewing sarcoma progenitor. Our data demonstrate that major architectural features of nuclear organization in cancer cells can depend on single oncogenes and are readily reversed to reestablish latent differentiation programs.

Screening for DAX1/EWS-FLI1 functional inhibitors identified dihydroorotate dehydrogenase as a therapeutic target for Ewing's sarcoma.

EWS-FLI1 is the most common oncogenic fusion protein in Ewing's sarcoma family tumors (ESFTs). DAX1, an orphan member of the nuclear receptor superfamily, is up-regulated by EWS-FLI1 and plays a key role in the transformed phenotype of ESFTs. To discover a functional inhibitor of DAX1 and EWS-FLI1, we screened small-molecular inhibitors using a DAX1 reporter assay system. K-234 and its derivatives, which were dihydroorotate dehydrogenase (DHODH) inhibitors, showed inhibitory effects in the reporter assay. K-234 inhibited the growth of Ewing's sarcoma with various fusion types, and K-234 derivatives altered the expression of EWS-FLI1-regulated genes. The DAX1 expression had no effect on the growth inhibitory effect of the K-234 derivatives, while DHODH overexpression or uridine treatment attenuated their inhibitory effects, suggesting that inhibition by K-234 derivatives occurs through DHODH inhibition. An in vivo study showed that a K-234 derivative clearly inhibited tumor growth in an Ewing's sarcoma xenograft mouse model. Taken together, the present results suggest that DHODH inhibitors can inhibit the function of DAX1/EWS-FLI1 in ESFTs and might be a therapeutic agent with potent anti-tumor activity for Ewing's sarcoma patients.

Oncogenic driver FGFR3-TACC3 requires five coiled-coil heptads for activation and disulfide bond formation for stability.

FGFR3-TACC3 represents an oncogenic fusion protein frequently identified in glioblastoma, lung cancer, bladder cancer, oral cancer, head and neck squamous cell carcinoma, gallbladder cancer, and cervical cancer. Various exon breakpoints of FGFR3-TACC3 have been identified in cancers; these were analyzed to determine the minimum contribution of TACC3 for activation of the FGFR3-TACC3 fusion protein. While TACC3 exons 11 and 12 are dispensable for activity, our results show that FGFR3-TACC3 requires exons 13-16 for biological activity. A detailed analysis of exon 13, which consists of 8 heptads forming a coiled coil, further defined the minimal region for biological activity as consisting of 5 heptads from exon 13, in addition to exons 14-16. These conclusions were supported by transformation assays of biological activity, examination of MAPK pathway activation, analysis of disulfide-bonded FGFR3-TACC3, and by examination of the Endoglycosidase H-resistant portion of FGFR3-TACC3. These results demonstrate that clinically identified FGFR3-TACC3 fusion proteins differ in their biological activity, depending upon the specific breakpoint. This study further suggests the TACC3 dimerization domain of FGFR3-TACC3 as a novel target in treating FGFR translocation driven cancers.

High-Grade Endometrial Stromal Sarcomas With YWHAE::NUTM2 Gene Fusion Exhibit Recurrent CDKN2A Alterations and Absence of p16 Staining is a Poor Prognostic Marker

High-grade endometrial stromal sarcomas (HGESSs) are aggressive uterine tumors harboring oncogenic fusion proteins. We performed a molecular study of 36 HGESSs with YWHAE::NUTM2 gene fusion, assessing co-occurring genetic events, and showed that these tumors frequently harbor recurrent events involving the CDKN2A locus on chromosome 9p. Using array-based copy number profiling and CDKN2A fluorescence in situ hybridization, we identified homozygous and hemizygous deletions of CDKN2A in 18% and 14% of tumors (n = 22 analyzed), respectively. While all YWHAE-rearranged HGESSs with retained disomy for CDKN2A were immunohistochemically positive for p16INK4 (p16), all tumors with homozygous deletion of CDKN2A showed complete absence of p16 staining. Of the 2 tumors with a hemizygous deletion of CDKN2A, 1 showed diffuse and strong p16 positivity, whereas the other showed complete absence of staining. In the p16-negative case, we did not find intragenic mutations or DNA promoter methylation to explain the p16 protein loss, implicating other mechanisms in the regulation of protein expression. In our cohort, subclonal or complete absence of p16 staining was associated with worse overall survival compared with positive p16 staining (1-year overall survival: 28.6% vs 90.7%, respectively; n = 32; P < .001), with all 7 patients in the p16-negative group having succumbed to their disease within 2 years of diagnosis. Our results suggested CDKN2A alterations as a cooperative driver of tumorigenesis in a subset of HGESSs with the YWHAE::NUTM2 gene fusion and showed p16 to be a potential prognostic marker.

Exploration of Flavonoids as Lead Compounds against Ewing Sarcoma through Molecular Docking, Pharmacogenomics Analysis, and Molecular Dynamics Simulations.

Ewing sarcoma (ES) is a highly malignant carcinoma prevalent in children and most frequent in the second decade of life. It mostly occurs due to t(11;22) (q24;q12) translocation. This translocation encodes the oncogenic fusion protein EWS/FLI (Friend leukemia integration 1 transcription factor), which acts as an aberrant transcription factor to deregulate target genes essential for cancer. Traditionally, flavonoids from plants have been investigated against viral and cancerous diseases and have shown some promising results to combat these disorders. In the current study, representative flavonoid compounds from various subclasses are selected and used to disrupt the RNA-binding motif of EWS, which is required for EWS/FLI fusion. By blocking the RNA-binding motif of EWS, it might be possible to combat ES. Therefore, molecular docking experiments validated the binding interaction patterns and structural behaviors of screened flavonoid compounds within the active region of the Ewing sarcoma protein (EWS). Furthermore, pharmacogenomics analysis was used to investigate potential drug interactions with Ewing sarcoma-associated genes. Finally, molecular dynamics simulations were used to investigate the stability of the best selected docked complexes. Taken together, daidzein, kaempferol, and genistein exhibited a result comparable to ifosfamide in the proposed in silico study and can be further analyzed as possible candidate compounds in biological in vitro studies against ES.

Current State of Immunotherapy and Mechanisms of Immune Evasion in Ewing Sarcoma and Osteosarcoma

We argue here that in many ways, Ewing sarcoma (EwS) is a unique tumor entity and yet, it shares many commonalities with other immunologically cold solid malignancies. From the historical perspective, EwS, osteosarcoma (OS) and other bone and soft-tissue sarcomas were the first types of tumors treated with the immunotherapy approach: more than 100 years ago American surgeon William B. Coley injected his patients with a mixture of heat-inactivated bacteria, achieving survival rates apparently higher than with surgery alone. In contrast to OS which exhibits recurrent somatic copy-number alterations, EwS possesses one of the lowest mutation rates among cancers, being driven by a single oncogenic fusion protein, most frequently EWS-FLI1. In spite these differences, both EwS and OS are allied with immune tolerance and low immunogenicity. We discuss here the potential mechanisms of immune escape in these tumors, including low representation of tumor-specific antigens, low expression levels of MHC-I antigen-presenting molecules, accumulation of immunosuppressive M2 macrophages and myeloid proinflammatory cells, and release of extracellular vesicles (EVs) which are capable of reprogramming host cells in the tumor microenvironment and systemic circulation. We also discuss the vulnerabilities of EwS and OS and potential novel strategies for their targeting.

Structural insights into p300 regulation and acetylation-dependent genome organisation

Histone modifications are deposited by chromatin modifying enzymes and read out by proteins that recognize the modified state. BRD4-NUT is an oncogenic fusion protein of the acetyl lysine reader BRD4 that binds to the acetylase p300 and enables formation of long-range intra- and interchromosomal interactions. We here examine how acetylation reading and writing enable formation of such interactions. We show that NUT contains an acidic transcriptional activation domain that binds to the TAZ2 domain of p300. We use NMR to investigate the structure of the complex and found that the TAZ2 domain has an autoinhibitory role for p300. NUT-TAZ2 interaction or mutations found in cancer that interfere with autoinhibition by TAZ2 allosterically activate p300. p300 activation results in a self-organizing, acetylation-dependent feed-forward reaction that enables long-range interactions by bromodomain multivalent acetyl-lysine binding. We discuss the implications for chromatin organisation, gene regulation and dysregulation in disease.

Preclinical In Vivo Activity of the Menin Inhibitor Ziftomenib (KO-539) in Pediatric KMT2A-Rearranged Acute Lymphoblastic Leukemia

Background: Chemotherapy resistance and subsequent relapse remain a major cause of childhood cancer mortality, particularly for infants (<12 months of age) with KMT2A-rearranged (R) B-acute lymphoblastic leukemia (B-ALL), who have extremely poor clinical outcomes with event-free survival <20% for highest-risk patients. KMT2A (MLL1) is a histone lysine N-methyltransferase involved in HOX-family and MEIS1 gene regulation that is commonly rearranged with other gene partners to form oncogenic KMT2A fusion proteins that drive acute lymphoid or myeloid leukemogenesis. Recent preclinical and early clinical studies have reported successful targeting of the menin scaffold protein within KMT2A fusion complexes in adults with KMT2A-R acute leukemias (Wang ASH 2020 abstract #115, Stein ASH 2021 abstract #699), but minimal data in pediatric patients exist to date. We hypothesized that the selective menin inhibitor ziftomenib (KO-539) would have potent activity in preclinical patient-derived xenograft (PDX) models of infant and non-infant pediatric KMT2A-R ALL and could enhance chemosensitivity in these often-chemoresistant leukemias. Methods: To test these hypotheses, we first measured the ability of ziftomenib to inhibit in vitro viability and KMT2A-menin complex protein targets in human KMT2A-R and KMT2A wild-type ALL cell lines in vitro using Cell Titer Glo and immunoblotting assays, respectively. We then assessed the ability of ziftomenib as monotherapy or in combination with chemotherapy to inhibit in vivo leukemia proliferation in PDX models of de novo or relapsed KMT2A-R ALL harboring various fusion partners (n=8; Loftus Haematologica 2021) after drug dose-finding in a bioluminescent KMT2A-R ALL cell line xenograft model. NSG mice engrafted with human KMT2A-R ALL cells were randomized to treatment with vehicle, ziftomenib 150 mg/kg via oral gavage (PO) daily x 5 days/week, vincristine 1 mg/kg intraperitoneally once weekly, dexamethasone 1 mg/kg PO daily x 5 days/week, ziftomenib + vincristine, or ziftomenib + vincristine for 3-6 weeks depending on rate of ALL progression in control animals necessitating study termination. Human CD45+ CD19+ ALL cell counts were measured weekly via retro-orbital venous bleeding and in end-study murine spleens via quantitative flow cytometry analysis as previously described (Tasian Blood 2017a, Hurtz JCI 2020). Results: We observed preferential in vitro sensitivity to ziftomenib of KMT2A-R ALL cell lines HB11;19 (half-maximal inhibitory concentration [IC50] 11.1 nM), KOPN-8 (IC50 49.1 nM), and SEM (IC50 32.6 nM) versus KMT2A wild-type NALM-6 cells (IC50 400.8 nM). Ziftomenib treatment of infant, non-infant pediatric, and young adult KMT2A-R ALL PDX models (comprised of more common KMT2A-AFF1 and KMT2A-MLLT1 rearrangements or other less common KMT2A fusions) induced significant inhibition of in vivo leukemia proliferation compared to vehicle treatment. Despite the remarkable potency of single-agent ziftomenib in all models tested, combination of ziftomenib with vincristine or dexamethasone further enhanced reduction of human ALL disease burden in end-study murine spleens, even in aggressive relapsed infant ALL PDX models (representative data in Figure 1). Conclusions: Our data demonstrate in vitro target inhibition and remarkable in vivo activity of the menin inhibitor ziftomenib against infant, pediatric, and young adult KMT2A-R ALL. Ziftomenib monotherapy activity was further augmented in most models when combined with conventional chemotherapy drugs vincristine or dexamethasone, which were well-tolerated in PDX mice. Based upon early clinical safety/tolerability data for ziftomenib in adult patients with acute leukemias and our pediatric-specific data described herein, a phase 1 clinical trial of ziftomenib in combination with multi-agent chemotherapy for children with relapsed/refractory KMT2A-rearranged, NUP98-rearranged, or NPM1-mutant acute leukemias is planned. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Establishment and Characterization of a Model of Acquired Resistance to DOT1L Inhibition in KMT2A-Rearranged Acute Lymphoblastic Leukemia Cells

KMT2A-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year of age) is an aggressive malignancy with event-free survival rates of only ~37%. KMT2A translocation represents the cytogenetic hallmark of this type of leukemia that is largely driven by inappropriate epigenetic events: oncogenic KMT2A fusion proteins recruit the histone methyltransferase DOT1L which leads to misplaced H3K79 methylation inducing an abnormal transcriptomic landscape that favors leukemia development. Therefore, the development and subsequent (pre-)clinical testing of the specific DOT1L inhibitor pinometostat was anticipated to be key to the successful treatment of KMT2A-rearranged acute leukemia. However, although clinical trials in adult KMT2A-rearranged acute leukemia patients did report initial responses (including complete remissions), disease progression due to treatment-related resistance appeared unavoidable. Nonetheless, while these observations urge the development of more effective inhibitors of DOT1L, the mechanisms underlying acquired resistance to DOT1L inhibition may well provide valuable insights into the unique biology of KMT2A-rearranged leukemia. To explore this, we provoked and established acquired pinometostat resistance in a pediatric KMT2A::AFF1+ ALL cell line model that stably became ~35-fold more resistant to DOT1L inhibition. Interestingly, pinometostat was still able to completely inhibit H3K79 di-methylation (H3K79me2) but failed to induce apoptosis, indicating that these cells largely became independent of DOT1L-mediated H3K79 methylation. Yet, shRNA-mediated knock-down of KMT2A::AFF1 revealed that these cells remained completely dependent on the presence of the KMT2A-fusion protein. In addition, pinometostat-resistant KMT2A-rearranged ALL cells proliferate more slowly and appeared to have switched their preference from oxidative phosphorylation to glycolysis to provide energy for metabolic activities, while remaining perfectly viable. As anticipated, pinometostat-resistant cells displayed a slight cross-resistance to most chemotherapeutic agents currently used in ALL treatments but became remarkably more sensitive toward the BCL-2 inhibitor venetoclax. To further characterize these cells and gain insights into the underlying mechanisms of resistance, we performed RNA-, ATAC-, and ChIP-sequencing analysis of KMT2A and AFF1 as well as of histone marks essential in KMT2A-rearranged ALL, including H3K79me2, H3K4 tri-methylation (H3K4me3) and H3K27 acetylation (H3K27ac). This revealed that acquired resistance to DOT1L inhibition leads to a selective loss of KMT2A-fusion driven epigenetic regulation and expression of genes such as PROM1 (encoding the hematopoietic/ leukemia stem cell marker CD133) and its enhancer TAPT1, as well as putative KMT2A::AFF1 target genes including RUNX2, GNAQ, SERPINB1, PRSS12, and ZC3H12C. In contrast, however, the expression of (and levels of H3K79 methylation at) other established KMT2A::AFF1 target genes like HOXA9, HOXA10, MEIS1, and CDK6 remained unaffected. Concomitantly, pinometostat-resistant KMT2A-rearranged ALL cells are characterized by significant enrichment of gene expression of various MYC targets, and specific up-regulation of genes associated with a myeloid immunophenotype, including CD33, CD38, LILRB4, MPEG1, LIMK1, and CCL5. Taken together, we here present an in vitro model of acquired resistance to DOT1L inhibition in KMT2A-rearranged ALL, instantly providing new insights and raising important questions regarding this elusive type of leukemia. For instance, KMT2A-rearranged ALL cells readily adapt to prolonged inhibition of H3K79 methylation but remain dependent on the KMT2A fusion, suggesting that recruitment of DOT1L may not be the only oncogenic action exerted by KMT2A fusion proteins. Also, our model demonstrates that under prolonged pressure of DOT1L inhibition, KMT2A-rearranged ALL cells seem to initiate a reprogramming process that involves the acquirement (or selection) of myeloid-like characteristics, an ability that may be connected to leukemic lineage switches which are not uncommon in KMT2A-rearranged acute leukemias. Hence, our model represents an important tool to study the complex biology of KMT2A-rearranged leukemia, and its existence and availability requires to be shared with the community.

CBFA2T3-GLIS2 model of pediatric acute megakaryoblastic leukemia identifies FOLR1 as a CAR T cell target.

The CBFA2T3-GLIS2 (C/G) fusion is a product of a cryptic translocation primarily seen in infants and early childhood and is associated with dismal outcome. Here, we demonstrate that the expression of the C/G oncogenic fusion protein promotes the transformation of human cord blood hematopoietic stem and progenitor cells (CB HSPCs) in an endothelial cell coculture system that recapitulates the transcriptome, morphology, and immunophenotype of C/G acute myeloid leukemia (AML) and induces highly aggressive leukemia in xenograft models. Interrogating the transcriptome of C/G-CB cells and primary C/G AML identified a library of C/G-fusion-specific genes that are potential targets for therapy. We developed chimeric antigen receptor (CAR) T cells directed against one of the targets, folate receptor α (FOLR1), and demonstrated their preclinical efficacy against C/G AML using in vitro and xenograft models. FOLR1 is also expressed in renal and pulmonary epithelium, raising concerns for toxicity that must be addressed for the clinical application of this therapy. Our findings underscore the role of the endothelial niche in promoting leukemic transformation of C/G-transduced CB HSPCs. Furthermore, this work has broad implications for studies of leukemogenesis applicable to a variety of oncogenic fusion-driven pediatric leukemias, providing a robust and tractable model system to characterize the molecular mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy.