Background: Patients with Acute Myeloid Leukemias (AML) harboring MLLT10 (AF10) fusion oncogenes have a 5-year survival rates of <10%. The PICALM-MLLT10 (CALM-AF10) fusion is found in 1-2% of AMLs and is associated with a poor prognosis. These leukemiasharbor increased HOXA gene expression, similar to leukemias with Lysine Methyltransferase 2A (KMT2A) gene rearrangements. We have recently shown that PICALM-MLLT10 activates the homeobox gene SIX1 in addition to HOXA genes. SIX1, together with its cofactor Eyes Absent 2 (EYA2), a protein tyrosine phosphatase, is involved in cell proliferation and embryogenesis and transcriptionally activates developmental genes. Overexpression of SIX1 has been observed in mesenchymal and epithelial malignancies including breast, ovarian, and esophageal cancers, in addition to being involved in accelerating the epithelial mesenchymal transition (EMT) and metastasis. The present studies evaluate the importance of SIX1 and its interaction with EYA2 in leukemogenesis, as well as the use of small molecule inhibitor in both in vitro and in vivo studies. Methods/Results: We first determined that increased SIX1 expression is associated with worse event free survival (EFS) and overall survival, while EYA2 correlates solely with EFS using the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database, which exploits a multiomic approach to evaluate profiles of multiple cancers. We next queried the Broad Institute Cancer Dependency Map and identified increased SIX1 expression in SHI-1 and OCI-M2 AML cell lines; neither displayed increased EYA2 expression. SIX1 expression in these cells was validated by immunoblot, and shRNA knockdown of SIX1 reduced proliferation in both cell lines. Overexpression of wild-type SIX1 in hematopoietic stem cells (HSCs) resulted in immortalization (tertiary colony formation in methylcellulose), but overexpression of a SIX1 mutant that is unable to bind EYA2 did not result in immortalization, suggesting that EYA proteins are required for SIX1 activity. Based on these observations, we evaluated the effects of EYA2 inhibition by a small molecule inhibitor of the EYA2 protein tyrosine phosphatase (LG1-34) along with an inactive analog (LG1-137) as a negative control (PID:38861151). CellTiterGlo assays measuring proliferation 72 hours after treatment established that the IC50 of LG1-34 for SHI-1 and OCI-M2 was 0.952 µM and 58.2 µM respectively, both showing over 10-fold increase in IC50 for the inactive compound LG1-137. LG1-34, but not LG1-137, showed a dose response effect on SHI-1 cell proliferation with 91%, 68%, 46%, 3%, and 2% viability at doses of 0.1 µM, 0.5 µM, 2.5 µM, 5 µM, and 10 µM, respectively. A similar, but not as robust, dose response effect was seen in OCI-M2 cells, with cell proliferation 99%, 68%, 41%, 20%, and 11% viability at doses of 0.1 µM, 0.5 µM, 2.5 µM, 5 µM, and 10 µM, respectively, corresponding with the IC50values obtained. IV administration of LG1-34 yielded a half-life of LG1-34 following PO administration (1.63 h) is more than twice that for IV administration (0.72 h), while higher plasma (2.8 µM) and brain (2.8 µM) concentrations were obtained with IV compared with PO administration (1.3 µM plasma and 0.8 µM brain). Daily treatment with LG1-34 for 28 d showed no showed no changes in weight or perturbations of CBC parameters. Daily treatment of NSG mice transplanted with SHI-1leukemia cells with 100 mg/kg of LG1-34 (n=5) prolonged survival by over 10 days (p= 0.0254). Discussion: AMLs harboring a MLLT10 translocation are associated with a poor prognosis. We have shown that a PICALM-MLLT10 fusion expresses the homeobox gene SIX1, which is also associated with worse outcomes. We have demonstrated that SIX1 requires EYA2 for immortalization of HSCs, and that treatment with a small molecule inhibitor of EYA2 reduces proliferation of AML cells in vitro and prolongs survival of leukemia-bearing mice in vivo. The results suggest an important role for SIX1/EYA2 in myeloid leukemogenesis, and the potential for EYA2 inhibition as a novel therapeutic modality.

Background: The CALM-AF10 (PICALM-MLLT10) fusion, found in 5-10% of pediatric T-ALLs (T-cell acute lymphoblastic leukemias), is associated with a poor prognosis. CALM-AF10 leukemias, which are dependent on the nuclear export protein XPO1, share many traits with other poor prognosis leukemias expressing translocations or rearrangements of the Lysine Methyltransferase 2A (KMT2A) gene, including aberrant activation of HOXA genes. In addition to HOXA gene activation, we previously demonstrated that CALM-AF10 also activates the homeobox gene SIX1. Together with its tyrosine phosphatase cofactor Eyes Absent 2 (EYA2), SIX1 transcriptionally activates developmental genes involved in cell proliferation and embryogenesis. SIX1 overexpression has been observed in mesenchymal and epithelial malignancies (e.g. breast, ovary, esophagus, gliomas), and SIX1 is involved in accelerating the epithelial mesenchymal transition and metastasis. The present studies evaluate the importance of SIX1 and its interaction with EYA2 in leukemogenesis, using hematopoietic stem cells and established leukemia cell lines. In addition, we assess the role of small molecule inhibitors of EYA2 in leukemia cells in vitroand in vivo. Methods/Results: Using the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database, which exploits a multiomic approach to evaluate profiles of multiple cancers, we established that increased SIX1 expression in relapsed T-ALL patients is associated with a worse event free survival (EFS) and overall survival (OS), while increased EYA2 expression is associated with worse EFS. We used the Broad Institute Cancer Dependency Map to identify that Jurkat leukemia cells, developed from a 14-year-old patient with T-ALL, had increased expression of SIX1, but not EYA2. RT-qPCR and immunoblot validated increased SIX1 expression. Compared to unaltered Jurkat leukemia cell lines, full CRISPR-Cas9 knockout of SIX1 abrogatedand partial knockout of SIX1 attenuated proliferation of Jurkat cells. To gain insight into the SIX1/EYA2 interaction, we assessed the immortalization potential of fetal liver hematopoietic stem cells (FL-HPs) transduced with a wild-type SIX1 and a mutant of SIX1 unable to bind EYA2 (SIX1EYA) through serial replating in methylcellulose colony assays. Wild-type SIX1, but not SIX1EYA, was able to immortalize FL-HPs, indicating that EYA proteins are required for SIX1-dependent immortalization. Since SIX1 expression is increased in Jurkat cells, and EYA2 is required for SIX1-induced immortalization of FL-HPs, we hypothesized that inhibition of EYA2 would slow the proliferation of SIX1-expressing Jurkat cells. We used two small molecule inhibitors of EYA2 phosphatase, 9987 and LG1-34 (PID:38861151); LG1-137, an analog of LG1-34 that lacks the tyrosine phosphatase activity of LG1-34, was used as a negative control. CellTiterGlo assays measuring proliferation 72 hours after treatment established that the IC50 for 9987 in Jurkat cells was 33.1µM, and that the IC50 of LG1-34 and LG1-137 was 0.74µM and 58.2µM respectively. LG1-34, but not LG1-137, showed a dose response effect on Jurkat leukemia cell proliferation with 99%, 68%, 41%, 20%, and 11% viability at doses of 0.1µM, 0.5µM, 2.5µM, 5.0µM, and 10µM, respectively. Intriguingly, addition of the XPO1 inhibitor KPT-330 to LG1-34 potentially enhances the inhibitory ability of LG1-34. In vivo pharmacokinetics studies demonstrated that IV administration of LG1-34 resulted in higher plasma (2.8µM) and brain (2.8µM) concentrations compared with PO administration (1.3µM plasma and 0.8µM brain). In addition, the half-life of LG1-34 following PO administration (1.63h) is more than twice that for IV administration (0.72h). Safety studies showed no changes in weight or perturbations of CBC parameters following 28 days of LG1-34 treatment. Daily treatment of NSG mice transplanted with Jurkatleukemia cells with 100 mg/kg of LG1-34 (n=5) prolonged survival by over 10 days (p= 0.0075).Discussion: SIX1 overexpression is associated with worse prognosis in T-ALL as well as other leukemias. As SIX1 requires EYA2 for immortalization of FL-HPs, we assessed the efficacy of available small molecule inhibitors of EYA2 to interfere with proliferation of SIX1-expressing leukemia cell lines. The ability of 9987 and LG1-34 to impair proliferation of SIX1-expressing leukemias both in vitro and in vivo supports a role for SIX1/EYA2 in these leukemias.

Juvenile myelomonocytic leukemia (JMML), a clonal hematologic malignancy, originates from mutated hematopoietic stem cells (HSCs). The mechanism sustaining the persistence of mutant stem cells, leading to leukemia development, remains elusive. In this study, we conducted comprehensive examination of gene expression profiles, transcriptional factor regulons, and cell compositions/interactions throughout various stages of tumor cell development in Ptpn11 mutation-associated JMML. Our analyses revealed that leukemia-initiating Ptpn11 E76K/+ mutant stem cells exhibited de novo activation of the myeloid transcriptional program and aberrant developmental trajectories. These mutant stem cells displayed significantly elevated expression of innate immunity-associated anti-microbial peptides and pro-inflammatory proteins, particularly S100a9 and S100a8. Biological experiments confirmed that S100a9/S100a8 conferred a selective advantage to the leukemia-initiating cells through autocrine effects and facilitated immune evasion by recruiting and promoting immune suppressive myeloid-derived suppressor cells (MDSCs) in the microenvironment. Importantly, pharmacological inhibition of S100a9/S100a8 signaling effectively impeded leukemia development from Ptpn11 E76K/+ mutant stem cells. These findings collectively suggest that JMML tumor-initiating cells exploit evolutionarily conserved innate immune and inflammatory mechanisms to establish clonal dominance.

Introduction Acute lymphoblastic leukemia (ALL) with KMT2A-rearrangement (KMT2A-r) in infants <1 year is a high-risk childhood ALL subtype, with consistently poor event-free survival (EFS) of approximately 35% when treated with intensive chemotherapy with or without hematopoietic stem cell transplant. Infant ALL blasts are characterized by DNA hypermethylation, which is hypothesized to contribute to chemoresistance by altering transcriptional regulation of gene expression. In preclinical studies of KMT2A-r blasts, epigenetic priming with DNA methyltransferase inhibitors improved the in vitro cytotoxicity of chemotherapy. Azacitidine, a pyrimidine nucleoside analog of cytidine and hypomethylating agent, has been used in combination with chemotherapy in children with leukemia. We previously reported that azacitidine was safe and well tolerated in AALL15P1 (ASPHO 2021) and herein we report survival outcomes. Methods The Children's Oncology Group (COG) trial AALL15P1 (NCT02828358) was a single arm, open label, groupwide pilot trial. The primary aim of the trial was to evaluate the tolerability of azacitidine in addition to Interfant-06 standard chemotherapy in infants with newly diagnosed KMT2A-r ALL. Estimation of 5-year EFS was an exploratory aim, given the small sample size. Eligibility criteria included B-ALL or acute leukemia of ambiguous lineage with ≥50% B-lymphoblasts, <366 days of age at diagnosis, and >36 weeks gestational age at enrollment. Exclusions included Down syndrome, secondary ALL, and prior cytotoxic therapy (except intrathecal chemotherapy or corticosteroids). Following an Interfant-based induction, infants with KMT2A-r received 4 courses of azacitidine, 2.5 mg/kg/dose intravenously over 10-40 minutes daily for 5 consecutive days in each course, immediately preceding the start of a chemotherapy course on day 6. Infants without KMT2A-r were removed from protocol following induction and did not receive azacitidine. Treatment failure was defined as failure to achieve M1 marrow status (<5% blasts by morphology) with resolution of extramedullary leukemia by the end of consolidation. EFS and overall survival (OS) were measured from the time of enrollment. Results The study accrued from March 2017 to December 2019 and all protocol-directed treatment concluded in December 2021. Of the 78 infants enrolled, 56 had KMT2A-r (72%), and 53 completed induction therapy and received at least 1 course of azacitidine. No patients were ineligible. Diagnostic clinical characteristics of infants with KMT2A-r included four infants age <7 days (7%), 13 age <90 days (23%), 18 with white blood cell count ≥300,000/µL (32%), 32 CNS2 (57%), and four CNS3 (7%). As of the data cutoff (06/30/2022), the median follow-up is 3.8 years, and the 3-year EFS (SE) and OS (SE) rates are 34.2% (+/- 0.08) and 63.8% (+/-0.08), respectively, for infants with KMT2A-r. Six infants experienced treatment failure. Minimal residual disease (MRD) levels of marrow blasts by flow cytometry in COG-approved laboratories were submitted for 49 KMT2A-r patients at the end of induction. Of those, 32 were MRD negative <0.01% (65%), eight MRD positive 0.01%-<1% (16%) and nine MRD positive ≥1% (18%). Event-free survival was significantly associated with MRD; the 3-year EFS of patients with any positive MRD was 20.6% (+/-0.13) vs. 40.1% (+/-0.09) (p=0.0185) for those without MRD. As previously reported, at no time did the trial meet or exceed the pre-determined dose limiting toxicity boundaries, and the rates and types of toxicities observed were within the expected range for infants receiving standard chemotherapy alone. Conclusions Epigenetic priming with azacitidine prior to standard chemotherapy was well tolerated in infants with KMT2A-r ALL, but the EFS was consistent with the poor survival of historical outcomes. Positive flow MRD at the end of induction predicted a higher risk of treatment failure, relapse, or death, in comparison to negative MRD, but EFS was still unacceptably low for MRD-negative patients. There remains an urgent need for improved therapies for infants with KMT2A-r ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Background: Leukemia is the most common type of childhood cancer. Although the prognosis for many pediatric leukemias has improved, leukemias associated with the t(10;11) CALM-AF10 translocation remain difficult to treat. CALM-AF10 leukemias account for ~5-10% of childhood T-cell acute lymphoid leukemia (T-ALL) as well as a subset of acute myeloid leukemia (AML). CALM-AF10 leukemias exhibit increased expression of proleukemic HOXA genes, but relatively little is known about the cellular mechanisms that drive CALM-AF10 leukemogenesis. Our laboratory has demonstrated that the CALM protein contains a nuclear export signal (NES) that is critical for CALM-AF10-dependent leukemogenesis. The NES interacts with the CRM1/XPO1 nuclear export receptor, which shuttles proteins from the nucleus to the cytoplasm through the nuclear pore complex. We have shown that transcriptional activation of HOXA genes by CALM-AF10 is critically dependent on its interaction with CRM1. Importantly, CRM1 does not contain a recognized DNA binding domain, and it is not currently understood how the CALM-AF10/CRM1 complex interacts with regulatory regions of HOXAgenes. In order to identify proteins that mediate the interaction between the CALM-AF10/CRM1 complex and DNA, we took advantage of a proximity-based labeling approach using BioID2, a second-generation biotin ligase. When fused to a protein of interest and in the presence of biotin, BioID2 biotinylates proteins in close proximity to the ligase. These biotinylated proteins can then be identified by mass spectrometry (MS). Methods: We prepared an expression plasmid in which BioID2 was cloned in-frame with CALM-AF10. We then transiently transfected Human Embryonic Kidney 293 (HEK293) cells with the BioID2-CALM-AF10 plasmid, grew them in the presence or absence of biotin, and performed streptavidin-pulldown followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to identify candidate interacting proteins. Proteins were considered candidates if they had a peptide spectrum match (PSM) score > 10 and at least a two-fold greater PSM score versus negative control. We validated direct interactions of candidate proteins with CALM-AF10 by performing co-immunoprecipitation experiments. Results: We first confirmed that the addition of BioID2 to CALM-AF10 does not affect the transcriptional activation of HOXA genes or CALM-AF10 mediated immortalization of hematopoietic stem cells. We carried out three independent transfections/LC-MS/MS experiments, which identified 71, 95 and 61 proteins, respectively. Of the proteins identified, 11 candidates were common to all three experiments.Of particular interest, we identified Disruptor Of Telomeric silencing 1-Like (DOT1L), a protein known to interact with AF10, and Nuclear pore complex protein 214 (NUP214), a protein that has been identified in leukemogenic translocations. The nine additional candidate proteins included: EPS15, DVL2, DVL3, and DDX3X -all known to play a role in leukemogenesis. We performed initial validation of direct interactions via co-immunoprecipitation and found that Epidermal Growth Factor Receptor Substrate 15(EPS15) co-precipitates with CALM-AF10. Conclusion: We used biotin ligase-dependent proximity-based labeling to identify candidate proteins that potentially interact with the CALM-AF10 fusion protein. Our identification of DOT1L validates the approach, since DOT1L is known to interact with CALM-AF10. We have started to investigate other candidate proteins, focusing on known translocation partners in various leukemias. Our screen identified EPS15, a protein involved in receptor-mediated endocytosis of epidermal growth factor and a known translocation partner for MLL/KMT2A. KMT2A-EPS15 translocations (t(1;11)(p32;q23)) have been identified in both AML and ALL, and KMT2A-EPS15 is among the eight most common KMT2A rearrangements. We have shown that EPS15 co-immunoprecipitates with CALM-AF10, suggesting that EPS15 may also play a role in CALM-AF10 leukemogenesis. Further evaluation of this interaction is underway, and may lead to identification of novel pathways involved in CALM-AF10 leukemogenesis. Disclosures No relevant conflicts of interest to declare.

Background : The CALM-AF10 translocation is found 5-10% of T-cell acute lymphoblastic leukemias (T-ALL), and a subset of acute myeloid leukemias (AML). CALM-AF10 leukemias are characterized by elevated expression of proleukemic HOXA genes. Since HOXA genes are difficult to target, we hypothesized that identification of non-HOXA CALM-AF10 effector genes could potentially yield novel therapeutic targets. To discover novel CALM-AF10-regulated genes, we took advantage of our prior observation that the nuclear export factor CRM1/XPO1 tethers CALM-AF10 to HOXA genes by interacting with a nuclear export signal within CALM. Using microarrays, we identified a set of genes that showed decreased expression in response to the CRM1 inhibitor, Leptomycin B (LMB), similar to Hoxa genes, in murine CALM-AF10 leukemia cells. Then using RNA-sequencing, we discovered a set of genes increased in murine hematopoietic stem cells transduced with CALM-AF10. There were 11 genes that were both decreased in response to LMB and increased in response to CALM-AF10, which included the Hoxa gene cluster, as well as Six1. Similar to HOXA genes, SIX1 is a homeobox gene that is associated with embryogenesis and is quiescent post-embryologically. Additionally, SIX1 and its cofactor EYA2 have been found to be overexpressed in numerous solid tumors, and inhibitor of the SIX1/EYA2 complex has recently been described. While there is evidence of a role for SIX1 in solid tumors, its role in leukemias has not been explored. Objective: To evaluate the role of SIX1 in CALM-AF10 leukemias. Design/Methods: RT-qPCR and Chromatin Immunoprecipitation (ChIP) were performed using bone marrow progenitors transduced with CALM-AF10 or an empty vector, with and without LMB. Methylcellulose colony assays assessed the ability of SIX1 to enhance self-renewal of hematopoietic progenitors. An inhibitor of the Six1/Eya2 interaction (compound 8430) was used to evaluate cell proliferation. Downstream targets of Six1 were evaluated using RT-qPCR in CALM-AF10 cells treated with Six1/Eya2 inhibitor (8430). Results: RT-qPCR confirmed overexpression of SIX1 in CALM-AF10 leukemia cells, and showed decreased SIX1 expression in the presence of LMB. Furthermore, ChIP revealed that CALM-AF10 binds to the SIX1 gene locus. Overexpression of SIX1 in fetal liver progenitors was sufficient to increase self-renewal potential. The 8430 Six1/Eya2 inhibitor slowed cell growth in CALM-AF10 cells compared to cells treated with DMSO alone. Finally, downstream targets such as Slc2a1, Cdk2, and Cyclina2 were decreased in 8430-treated CALM-AF10 leukemia cells. Conclusions: The SIX1 homeobox gene is highly expressed during embryogenesis, and its expression is silenced post-embryogenesis. Through an initial unbiased screen, we discovered that Six1 may play a role in CALM-AF10 leukemogenesis. We have determined that Six1 expression is upregulated in the presence of CALM-AF10. Further, we have shown a potential oncogenic role for Six1, as it was able to increase the self-renewal potential of hematopoietic progenitors. The role of Six1 in CALM-AF10 leukemia is further supported by the ability of a SIX1/EYA2 inhibitor to slow the growth of CALM-AF10 leukemia cells and decrease the expression of downstream targets of SIX1. These observations suggest that Six1 plays a pathogenic role in leukemogenesis, and may be a novel therapeutic target in CALM-AF10 leukemias. Disclosures No relevant conflicts of interest to declare.

Abstract Introduction: The Food and Drug Administration (FDA) expanded access program uses a single-patient use (SPU) mechanism to provide patient access to investigational agents prior to FDA approval and outside clinical trials, in situations where no satisfactory or comparable therapy is available. Genomic profiling of both newly diagnosed and relapsed/refractory childhood cancer has increased in the last decade, resulting in identification of new drug targets for pediatric malignancies. Recently, a review of SPU use in adult and pediatric patients at a single institution (Feit, JAMA Oncology 2019) showed that a markedly higher percentage of pediatric patients receive access through SPUs compared with adult patients, identifying this as an important means of pediatric drug access. However, little is known about the pediatric use and efficacy of SPUs in children. The aim of this study is to examine the SPU experience in pediatric and adolescent and young adult (AYA) cancer patients—specifically obtained for targeted therapies at three large pediatric cancer centers. Methods: All therapeutic SPUs obtained between January 1, 2014 and January 1, 2019 were evaluated at the Aflac Cancer & Blood Disorders Center (Atlanta, GA), Cincinnati Children’s Hospital (Cincinnati, OH), and the Memorial Sloan Kettering Cancer Center (New York, NY). Data were collected on the type of malignancy, agents requested, and corresponding molecularly informed targets, if applicable. Results: A total of 61 SPUs were approved in the five-year period, with 34 (55%) of them specific for access to agents based on somatic tumor mutations identified by genomic profiling. Among the disease groups, SPUs were most often requested for tumors affecting the central nervous system (CNS) (48%), followed by solid tumors (bone, liver, and kidney tumors) (26%), hematologic malignancies (leukemia/lymphoma) (21%), and other rare tumors (5%). Kinase inhibitors were the most frequently requested agents in the genomically defined category (n=34), specifically, FGFR (fibroblast growth factor receptor) inhibitors followed by drugs targeting NTRK 1/2/3 (tropomyosin receptor kinase (Trk) receptors). Most patients within this genomically targeted group (18/34) are currently receiving therapy with these agents. Conclusions: We found that SPUs represent an important means of access to therapeutic agents in the pediatric and AYA populations, with more than half of all SPUs based on rearrangements identified by genomic profiling. A broad range of agents were requested across CNS, solid tumor, and hematologic types. Furthermore, more than half of the patients remain on their respective SPU-approved drug. We are currently performing a more detailed analysis of clinical responses, as well as a time analysis from SPU initiation to approval for all patients in this cohort. Establishment of this cohort of patients across institutions will serve as the basis for a formal registry of pediatric SPUs, which will enable us to study their use and efficacy over time. Citation Format: Himalee Sabnis, Benjamin Mizukawa, Julia Glade-Bender, Jason Fangusaro, Stephen Roberts, Chanta Whitlow, Marilyn Winchester, Maureen O’Brien, Laura Agresta, Brian Turpin, Daniel Wechsler, Sharon Castellino, Neerav Shukla. Targeted therapies for children and young adults with cancer: Single-patient use (SPU) experience at three large pediatric cancer programs [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A50.