Abstract Introduction: B-cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer. Cooperative group trials and targeted therapies have significantly improved survival rates in pediatric B-ALL; however, 10-20% of patients still relapse, with 5-year overall survival post relapse around 50%. CD19-directed therapies including chimeric antigen receptor T-cell therapy (CART) and blinatumomab have rescued multiply relapsed B-ALL patients. Limitations of blinatumomab include continuous infusion for 28 days and unexpected hospital visits. As blinatumomab moves into frontline therapy it is noted that CD19 escape is prevalent. Inotuzumab Ozogamacin (InO) is an antibody-drug conjugate targeting CD22 on leukemic blasts given as a once-weekly infusion over three weeks. Phase 3 trials of InO in adults with B-ALL showed improved remission rates compared to standard chemotherapy and led to FDA approval of InO for adults (2019) and children (2024). This study aimed to describe our single-center experience of InO in patients with relapsed/refractory B-ALL, many of whom were heavily pretreated. Methods: We identified all patients treated at Children's Healthcare of Atlanta from 1/1/2015-7/28/2025 with B-ALL who received at least one dose of InO after time of relapse/refractory status. Patient demographics, treatment pre- and post- InO, and vital status were abstracted from the electronic medical record and reported descriptively. Results: Twenty-two patients with relapsed/refractory B-ALL received InO. The mean age at diagnosis and initial InO infusion was 10.5 and 12.5 years, respectively (range: 11 months-17 years; 23 months-20 years). Two patients (9%) were ≥18 years of age at time of first InO infusion. At diagnosis, 8 (36.5%) patients had National Cancer Institute (NCI) Standard Risk B-ALL, 13 (59.1%) were NCI High Risk, and 1 (4.5%) had infant ALL. Overall, 17/22 (77.3%) achieved at least 1 complete remission (CR, range: 0-5) prior to InO therapy, while 5/22 (22.7%) were refractory (unable to achieve CR) to initial therapy. Nineteen patients (86.4%) received a full course of InO (0.8mg/m2 day 1, 0.5mg/m2 day 8 and 15), 2/22 (9.1%) had disease progression in peripheral blood after the second InO dose and stopped treatment, and 1/22 (4.5%) received lower doses of InO. CR with minimal residual disease (MRD) ≤0.01% by Multiparameter Flow Cytometry (MFC) was achieved after InO in 14/22 patients (63.6%); only those who received full dose InO achieved CR. Of the patients with MRD <0.01% after InO, 8/14 (57.1%) are still alive at a median time of 2.3 years. Definitive therapy included CART (n=2, 14.3%), bone marrow transplant (BMT; n=9, 64.3%), and CART followed by BMT (CART+BMT; n=3, 13.6%). Survivors after InO include patients receiving CART (2/2, 100%), BMT (5/9, 55.6%) and CART-BMT (1/3, 33%). All patients who died after BMT were in remission; causes of death were attributed to BMT toxicity with 1 patient having defibrotide-treated veno-occlusive disease (VOD). The CART-BMT patient died from progressive disease. Of those patients not achieving remission post InO, 7/8 (87.5%) died, with 6/8 (75%) receiving no further cure-directed therapy following InO. Of the remaining 2 patients not in CR after InO, 1 is alive post-CART and the other underwent CART+BMT but died due to complications of BMT including defibrotide treated VOD (in remission at time of death). Interestingly, t(1;19) was found in 5/22 (22.7%) patients, all of whom died, and accounted for the majority 4/6 (66.6%) of patients with progression after InO. Conclusions: This single institution study shows that full-dose InO leads to MRD negative CR in relapsed/refractory patients with B-ALL, serving as an effective bridge to subsequent therapy with CART, BMT, or CART+BMT. Over half of these patients are still alive, further demonstrating the use of InO as a bridge from disease to consolidative therapy. While VOD is a common concern after InO treatment, in our cohort only 2 patients had defibrotide-treated VOD, both following BMT. Further adverse event evaluation is ongoing. In our small cohort the t(1;19) translocation appears to be a negative prognostic indicator for response to InO; future studies should further investigate this association. Overall, these data show InO is a viable alternative to targeted salvage therapies and has the benefit of being able to treat CD22-positive, leukemia with decreased burden of delivery for patients and families during drug infusion.

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.

Bone marrow aspirations are pivotal for diagnosing and monitoring various hematological conditions, including cancers. However, a significant portion (10%-50%) of aspirations yield suboptimal or inadequate diagnostic material. The difficulty and scarcity of bedside adequacy assessment strategies further exacerbate the challenges in this procedure, which can consequently lead to delays in diagnosis and treatment, among other complications. To address this unmet clinical need, we apply deep UV microscopy, a real-time, low-cost, label-free molecular imaging technology that recapitulates the appearance of Giemsa stains. We present results from a prospective clinical study comprising 51 pediatric oncology patients, where the deep UV images of unstained bone marrow aspirate smears are evaluated and compared with the clinical standard of care (a hematopathologist inspection of the same slides after Giemsa staining). Results show that both real-time visual UV inspection and an automated classification algorithm applied to the unstained deep UV images achieve accurate adequacy assessment, with accuracies of 94.1% and 95.7%, respectively. Additionally, we demonstrate whole-slide imaging of bone marrow aspirate smears using a compact and low-cost deep UV microscope that is well suited for point-of-care use. Together, this work has significant implications for improving bone marrow aspirations and the clinical management of many hematological patients. (c) 2025 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Bone marrow aspiration procedures play an important role in the assessment of patients with blood and marrow diseases, including cancers. Evaluating the adequacy of aspirates, indicated by the presence of bony spicules, is crucial to ensure the procedural success and collection of relevant diagnostic material. Unfortunately, inadequate samples occur in approximately 50 % of cases, requiring patients to undergo repeat procedures. This is particularly problematic for pediatric patients who need to be anesthetized before each procedure. The current gold standard is hematopathologist examination of Giemsa-stained slides, which is time consuming and requires expensive biochemical reagents and trained technicians. Recently, Here we present a portable, LED-based UV microscope designed for real-time inspection of bone marrow aspirates. We discuss results from a clinical trial with pediatric oncology patients demonstrating excellent agreement between UV examination of unstained slides and ground truth pathologist examination of stained slides. Furthermore, we demonstrate whole slide imaging using a previously developed, compact UV microscopy system and automated spicule detection with deep neural networks to work towards point-of-care applications.

Background: SIX1 is a homeobox gene that is involved in cell proliferation, embryogenesis, and apoptosis. Together with its cofactor Eyes Absent 2 (EYA2), a protein phosphatase, SIX1 transcriptionally activates developmental genes, and is expressed at low levels post-embryologically. Overexpression of SIX1 has been observed in mesenchymal and epithelial malignancies including breast cancer, ovarian cancer, gliomas and esophageal cancer, and SIX1 is involved in accelerating the epithelial mesenchymal transition (EMT) and metastasis. SIX1 has also been implicated in MLL/KMT2A leukemias, although its precise leukemogenic role has not been defined. We have previously demonstrated that SIX1 expression is increased in CALM-AF10 leukemia cells. The present studies evaluate the importance of SIX1 and its interaction with EYA2 in leukemogenesis, using hematopoietic stem cells (HSC) and established leukemia cell lines. In addition, we assess the role of small molecule inhibitors of EYA2 in leukemia cells in vitro, and establish pharmacokinetic parameters in mice in vivo. Methods/Results: We used the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database, which exploits a multiomic approach to evaluate profiles of multiple cancers, to determine that increased SIX1 expression is associated with worse event free survival (EFS) and overall survival (OS) in relapsed T-cell Acute Lymphoblastic Leukemia (T-ALL), Ambiguous Lineage Acute Leukemias (ALAL), and Acute Myeloid Leukemias (AML). While increased EYA2 expression is associated with worse EFS in T-ALL patients, it correlates with improved OS in B-ALL, ALAL, and AML patients. To evaluate whether established leukemia cell lines display increased SIX1 or EYA2 expression, we queried the Broad Institute Cancer Dependency Map. We identified increased SIX1 expression in Jurkat (T-ALL), SHI-1 (AML), and OCIM2 (AML) leukemia lines, though none had increased expression of EYA2. SIX1 expression in these cells was validated by immunoblot, and shRNA knockdown of SIX1 reduced proliferation of all three cell lines. To determine the impact of SIX1 and its interaction with EYA2 on HSC immortalization, we overexpressed SIX1 in HSCs. Wild-type SIX1 immortalizes HSCs, while SIX1 bearing a mutation in its EYA binding domain does not immortalize, indicating that EYA proteins are required for SIX1-dependent immortalization. Using co-immunoprecipitation in SIX1-overexpressing leukemia cell lines, we confirmed that SIX1 and EYA2 directly interact. To interfere with SIX1 activity, we used two small molecule inhibitors of EYA2 phosphatase (9987 and the recently described LG1-34 (PID: 38861151)); LG1-137, an analog of LG1-34 that has decreased binding affinity for the EYA2 phosphatase, was used as a negative control. Treatment of two murine CALM-AF10 leukemia lines as well as the human leukemia cell lines Jurkat, SHI-1, and OCI-M2 with 9987 impaired proliferation at doses ranging from 20-40 μM. LG1-34, a more potent version of 9987, also impaired proliferation of all three cell lines at doses ranging from 0.1-5 μM. Importantly, LG1-137 had no effect on these cells, with IC50 concentrations 30-90 fold higher than LG1-34. Preliminary in vivo pharmacokinetics studies demonstrated that IV administration leads to higher concentration in plasma (2.8 μM) and brain (2.8 μM) compared with PO administration (1.3 μM for plasma and 0.8 μM for brain), while the half-life of LG1-34 following PO administration (1.63 h) is more than twice that for IV administration (0.72 h). Discussion: Overexpression of SIX1 is associated with worse prognosis in T-ALL, ALAL and AML. Since SIX1 requires EYA2 for immortalization of HSCs, 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 supports a role for SIX1/EYA2 in these leukemias. These studies provide a framework for evaluating the efficacy of EYA2 inhibitors in murine leukemias as well as PDX models of leukemia. Future studies will assess the effect of EYA2 overexpression on immortalization in vitro, and will evaluate the effectiveness of EYA2 inhibitors in PDX and murine leukemia models.

Chromosome Region Maintenance 1 (CRM1), also known as Exportin 1 (XPO1), is a protein that is critical for transport of proteins and RNA to the cytoplasm through the nuclear pore complex. CRM1 inhibition with small molecule inhibitors is currently being studied in many cancers, including leukemias, solid organ malignancies and brain tumors. We review the structure of CRM1, its role in nuclear export, the current availability of CRM1 inhibitors, and the role of CRM1 in a number of distinct cellular processes. A deeper understanding of how CRM1 functions in nuclear export as well as other cellular processes may allow for the development of additional novel CRM1 inhibitors.

Patients with DNA double-strand breakage repair disorders are at increased risk of malignancy which is often difficult to treat given underlying sensitivity to chemotherapy and radiotherapy, lending an important role to hematopoietic stem cell transplantation. The choice of conditioning regimen used must balance reducing risk of rejection with minimizing excessive toxicity from myeloablative chemotherapy or ionizing radiation. We describe successful engraftment following a nonmyeloablative hematopoietic stem cell transplantation in a patient with Ligase IV syndrome and numerous pretransplant complications including malignancy, cardiac failure, and secondary hemophagocytic lymphohistiocytosis. Congruent with prior reports, a reduced intensity regimen appears efficacious in Ligase IV syndrome patients.

Background: The CALM-AF10 translocation is found in 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 sought to identify non- HOXA effector genes through RNA sequencing and Microarray analyses, and showed that SIX1 expression is increased in CALM-AF10 leukemia cells. Like HOXA genes, SIX1 is a homeobox gene involved in cell proliferation, embryogenesis, and apoptosis. SIX1 interacts with the cofactor Eyes Absent 2 (EYA2), a protein phosphatase, to transcriptionally activate developmental genes, the TGFβ pathway, and the epithelial-mesenchymal transition (EMT). SIX1 expression is elevated during development but decreases with maturation. Intriguingly, overexpression of SIX1 has been observed in numerous malignancies including breast cancer, ovarian cancer, gliomas and esophageal cancer, and SIX1 is involved in accelerating the epithelial mesenchymal transition (EMT) and metastasis. Knockdown of either SIX1 or EYA2 has been shown to reverse these effects, and two novel inhibitors - one of the SIX1/EYA2 complex (Compound 8430, “8430”) and one of the EYA2 phosphatase (Compound 9987, “9987”) - have replicated the effects of SIX1 or EYA2 knockdown. Methods: SIX1 gene and protein expression were assessed in leukemia cell lines via RT-qPCR and Western Blot. SIX1 expression vectors were transduced into fetal liver hematopoietic stem cells (HSC) and immortalization was assessed using colony assays. CALM-AF10 leukemia cells lines were derived from irradiated mice transplanted with HSC retrovirally transfected with CALM-AF10 plasmids created in our lab. The established leukemia cell lines Jurkat, MOLT16, OCI-M2, TF-1, and SHI-1 were obtained from ATCC or DSMZ. shRNAs targeting SIX1 were lentivirally transduced into CALM-AF10 and SHI1leukemia cell lines. Cell-Titer-Glo assays and liquid culture were used to assess the effects of shRNAs, 8430, or 9987 on cell proliferation and cell growth. SIX1 gene expression in leukemias was determined from The Cancer Dependency Map from The Broad Institute using R. Results: We first validated RNAseq and Microarray studies, showing that CALM-AF10 induces SIX1 expression in murine fibroblasts and murine HSC, and localizes to the SIX1 locus. Furthermore, SIX1 overexpression was sufficient to immortalize murine fetal liver HSC (FL-mHSCs), implying a role in leukemic transformation. Multiple murine derived CALM-AF10 cell lines displayed increased SIX1 expression compared to fresh murine bone marrow and isolated HSCs. shRNA knockdown of SIX1 decreased viability of murine CALM-AF10 cells in culture. In addition, both the SIX1/EYA2 complex inhibitor (8430) and the EYA2 inhibitor (9987) decreased CALM-AF10 leukemia cell proliferation in vitro. To expand the relevance of SIX1 to other leukemias, we then queried The Cancer Dependency Map from the Broad Institute and identified other leukemia cell lines with increased SIX1 expression, including Jurkat and Molt16 (T-ALL), and SHI-1, OCIM2, and TF-1 (AML). SIX1 expression in these cells was validated by immunoblot and shRNA knockdown of SIX1 slowed the proliferation of SHI-1 cells. Finally, the 8430 and 9987 inhibitors reduced the proliferation of Jurkat, OCI-M2, and SHI-1 cells at doses similar to those used in CALM-AF10 leukemias. Conclusions: We have identified involvement of the SIX1/EYA2 axis in several different leukemias, including T-ALL and AML. Furthermore, we have shown that inhibitors of the SIX1/EYA2 interaction and of EYA2 reduce the proliferation of SIX1 expressing leukemias, suggesting a potential new therapeutic target in these leukemias.

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.

Abstract 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 lymphoblastic 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 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 HOXA genes. 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. Human Embryonic Kidney 293 (HEK293) cells were transiently transfected with BioID2-CALM-AF10 and grown in the presence or absence of biotin. MS was performed to identify candidate interacting proteins. We validated direct interactions of candidate proteins with CALM-AF10 using co-immunoprecipitation experiments in HEK293 cells transfected with a CALM-AF10 plasmid. We confirmed that candidate proteins are present in murine CALM-AF10 leukemia cells via Western blotting. In order to efficiently knockout (KO) candidate proteins, we have generated a human U937 cell line (which harbors a t(10;11) CALM-AF10 translocation) with a stable incorporated Cas9. To assess whether KO of EPS15, DVL2 or CTTN affects HOXA5 expression, we performed RT-qPCR in U937-Cas9 cells lines with confirmed KO. Results: We carried out three independent transfections/MS experiments, which identified 71, 95 and 61 proteins, respectively. Of the proteins identified, 12 candidates were common to all three experiments . Importantly, 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 interacts with CRM1 and that is involved in leukemogenic translocations. We chose EPS15, DVL2 and CTTN for further study, as each of these proteins plays a role in leukemogenesis. We performed initial validation of direct interactions via co-immunoprecipitation and found that all three proteins co-precipitate with CALM-AF10. Western blotting showed that all three proteins are expressed in a murine CALM-AF10 leukemia cell line. We effectively knocked out EPS15 protein expression in U937 cells, and showed that HOXA5 expression is reduced in the setting of EPS15 knockout. 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 three candidate proteins - EPS15, DVL2 and CTTN - all of which are involved in leukemogenic transformation. We have shown that EPS15, DVL2 and CTTN are expressed in murine CALM-AF10 leukemia cells and directly interact with the CALM-AF10 fusion protein. Knockout of EPS15 in U937 cells results in decreased HOXA5 expression, suggesting the importance of EPS15 in CALM-AF10 leukemogenesis. Evaluation of the roles of these proteins in leukemogenesis may lead to identification of novel pathways involved in CALM-AF10 leukemogenesis. Disclosures No relevant conflicts of interest to declare.