Integrated Transcriptomics and Proteomics Identifies Therapeutic Targets in Pediatric Acute Myeloid Leukemia

Acute myeloid leukemia (AML) remains a therapeutic challenge with high mortality rates despite intensive and myeloablative therapies. While immunotherapies targeting CD19 have yielded remarkable outcomes in acute lymphoblastic leukemia, identifying similar antigen therapeutic targets in AML remains a challenge due to inherent heterogeneity associated with AML and overlapping immunophenotypes with normal hematopoietic stem and myeloid cell populations. Transcriptional heterogeneity within pediatric AML has primarily been linked to underlying fusion. Therefore, we integrated large transcriptomics and proteomics datasets from AML and normal tissues to identify potential targets expressed in leukemias, but not in normal bone marrow or other normal tissue types. To identify candidate therapeutic targets in pediatric AML, we leveraged transcriptome sequencing data from bone marrow aspirates or peripheral blood collected from 1,481 children, adolescents, and young adults with AML at the time of diagnosis. Patients were enrolled on one of four Children's Oncology Group trials spanning the past three decades: CCG-2961, AAML03P1, AAML0531, and AAML1031. We also leveraged transcriptome sequencing from normal bone marrow (NBM) and normal CD34+ hematopoietic stem and progenitor cells (HSPCs) in order to exclude targets that are highly expressed during normal hematopoiesis. Finally, we performed additional filtering based on proteomic databases to exclude targets that lack membrane localization (Human Protein Atlas, UniProt, and Ensembl) or that are highly expressed on normal tissue types (Human Proteome Map, Human Protein Atlas, and Proteomics DB databases) (Figure 1A). First, we computed the log expression ratio between AMLs and NBM/HSCPs for all protein coding genes. We next selected genes expressed greater than a threshold of two standard deviations above the mean in 50% or more of AMLs (Figure 1B). Additionally, we further selected genes on the basis of differential gene expression and absolute expression thresholds. This analysis was repeated for our entire pediatric AML cohort and the following AML subtypes: RUNX1-RUNX1T1, CBFB-MYH11, KMT2A-MLLT3, KMT2A-MLLT10, KMT2A-MLLT4, KMT2A-ELL, KMT2A-MLLT1, KMT2A-MLLT11, NUP98-NSD1, NUP98-KDM5A, and CBFA2T3-GLIS2. Candidate therapeutic targets were filtered based on membrane localization and normal tissue expression using the aforementioned proteomics databases (Figure 1C and 1D). Based on this algorithm, we identified a nonzero number of candidate therapeutic targets for each of our pediatric AML subtypes (Figure 1D). Intriguingly, we found no overlap between targets identified in our pediatric, adolescent, and young adult cohort and a previous similar analysis performed in AMLs diagnosed in older patients (PMID 29017060). This study demonstrates that by combining our large transcriptomics dataset with pre-existing proteomics datasets, we are able to identify a collection of candidate therapeutic targets in pediatric AML. Importantly, zero targets were identified that were inclusive to all pediatric AMLs within our cohort, which underscores the transcriptional heterogeneity that our group and others have previously identified in pediatric AML. Future preclinical and clinical studies will need to account for this heterogeneity by prioritizing targets on the basis of underlying molecular alteration. Our study comprises a platform and dataset of candidate targets for further functional validation and/or immunotherapy targeting studies in pediatric AML. Figure 1 Figure 1. Disclosures Shaw: T-Cell and/or Gene Therapy for Cancer: Patents & Royalties.

Role and Mechanism of circ_0058063/miR-635 Axis in the Malignant Phenotype of Multiple Myeloma RPMI8226 Cells

Objective. This study aims to explore circ_0058063 effect on multiple myeloma cells malignant phenotype and its feasible mechanism. Methods. We selected 47 cases of multiple myeloma tissues and 47 cases of normal bone marrow tissues and then used RT-qPCR method to test circ_0058063 and miR-635 expression in the tissues. Myeloma cells RPMI8226 were transfected with si-circ_0058063, miR-635 mimic, and si-circ_0058063 + anti-miR-635, respectively. Then, we adopt CCK-8 method, flow cytometry method, and Transwell and western blot methods to detect the influences of knockdown of circ_0058063 or miR-635 overexpression on RPMI8226 cell proliferation, apoptosis, migration, and invasion and also Ki-67, Bax, Bcl-2, MMP-2, and MMP-9 protein expression. The dual luciferase reporter gene assay experiment proved that it has regulatory relationship between circ_0058063 and miR-635. Results. circ_0058063 expression of multiple myeloma was higher than that in normal bone marrow tissue ( P < 0.05 ), while miR-635 expression was lower than that in normal bone marrow tissue ( P < 0.05 ). Knockdown of circ_0058063 or overexpression of miR-635 could reduce proliferation capacity, migration, invasion cell quantities, and Ki-67, MMP-2, MMP-9, and Bcl-2 protein expression ( P < 0.05 ), while increasing apoptosis rate together with Bax protein expression ( P < 0.05 ). circ_0058063 targets to negatively regulate miR-635, while knocking down miR-635 reverses the influences of knocking down circ_0058063 on RPMI8226 proliferation, apoptosis, migration, and invasion. Conclusion. circ_0058063 expression increased in multiple myeloma tissues. Knocking down its expression may inhibit myeloma proliferation, migration, and invasion by targeting and upregulating miR-635 and also promote cell apoptosis. As for multiple myeloma treatment, circ_0058063/miR-635 may provide new molecular targets.

7 Tesla Chlorine (35Cl) and Sodium (23Na) MR Imaging of an Enchondroma

We demonstrated the feasibility of 7 Tesla sodium (23Na) and chlorine (35Cl) MRI of a solitary enchondroma. For this, we established dedicated sequences on a 7-Tesla whole-body system with the following key parameters for 35Cl MRI: TE/TR = 0.35/60 ms, TRO = 5 ms, α = 90°, Δx3 = (6 mm)3, 3 averages, Tacq = 30 min and for 23Na MRI: TE/TR = 0.4/101 ms, TRO = 10ms; α = 90°; Δx3 = (1.9 mm)3, 3 averages, Tacq = 30 min 18 s. The measured apparent Na+ concentration was 255 mmol/l and was approximately 7-fold higher than the apparent Cl– concentration with about 36 mmol/l. Additionally, repeated proton MRI examinations demonstrated constant but subtle growth (≈ 0.65 ml/year) over 14 years. In conclusion, enchondromas obviously have a high contrast-to-noise ratio when compared with the normal bone marrow in 23Na and 35Cl MRI, which may contribute to detection and differentiation in unclear or subtle cases. Key Points: Citation Format

Improved Survival of Leukemic Mice Treated with Sodium Caseinate in Combination with Daunorubicin without Toxicity

In recent years, low doses of chemotherapy have been resumed and explored for the treatment of acute myeloid leukemia. Thus, CPX-351, a dual-drug liposomal encapsulation of cytarabine and daunorubicin, was approved by the US Food and Drug Administration, to deliver a synergistic 5 : 1 molar drug ratio into leukemia cells to a greater extent than normal bone marrow cells and significantly enhance survival compared with conventional treatment in older and newly diagnosed AML patients, but overall survival rate remains low; therefore, the need for new therapeutic options continues. Sodium caseinate (SC), a salt of casein, the main milk protein, has cytotoxic effect in leukemia cell lines, but promotes proliferation of hematopoietic normal cells, while its administration in leukemic mice promotes survival for more than 40 days, but bone marrow surviving mice still harbour leukemic cells, but it is not known whether the combination with cytarabine or daunorubicin can improve survival without damaging normal hematopoietic cells. Here, it is shown that, in vitro, the combination of the IC25 of SC-cytarabine or SC-daunorubicin synergizes in the elimination of leukemic cells, with evident induction of apoptosis, while the proliferation of mononuclear cells of bone marrow is not affected. In leukemic mice, the combined administration of SC-daunorubicin or SC-cytarabine promotes the highest survival rate at 40 days; in addition, no autoproliferating cells were detected in the bone marrow of survivors of more than 60 days, evidence of eradication of leukemic cells, but only the bone marrow of mice treated with the SC-daunorubicin combination proliferated in the presence of interleukin-3, which shows that this combination is not toxic to normal bone marrow cells, thus emerging as a possible antileukemic agent.

Restricted Immunoglobulin Joining Chain (IgJ) Protein Expression in B Lymphoblastic Leukemia (B-ALL) Based on B-ALL Subtype

Background Philadelphia-like (Ph-like) B Lymphoblastic Leukemia (B-ALL) is a high-risk subtype of B-ALL that lacks the BCR-ABL1 fusion but has a gene expression profile similar to Philadelphia positive (Ph+) B-ALL. Gene expression profiling has previously identified Immunoglobulin Joining chain (IgJ) overexpression at the mRNA level in Ph-like B-ALL. This is surprising since IgJ is normally expressed by mature or maturing B cells. The normal function of IgJ protein is to concatenate monomers of immunoglobulin IgM or IgA into the mature pentameric and dimeric forms of these molecules respectively. IgJ also plays a crucial role in transport of IgA protein across mucosal epithelium to facilitate mucosal humoral immunity. In hematopathology, J chain immunohistochemistry (IHC) has been used to identify neoplastic cells in nodular lymphocyte predominate Hodgkin lymphoma (NLPHL) and can be used in distinguishing this disease from morphologic mimickers. It does not have known diagnostic utility outside of this context. Lymphoblasts do not typically express immunoglobulins at the protein level. Therefore, we sought to determine the protein expression of IgJ in B-ALL and to determine whether IgJ immunohistochemistry may be employed in identifying particular subtypes of B-ALL. Methods We selected a total of 46 B-ALL cases diagnosed from a bone marrow sample at our institution from 2016-2019 with adequate diagnostic material for IHC. This included 5 cases of Ph-like B-ALL, all with a CRLF2 rearrangement and overexpression, 7 de novo Ph+ B-ALL and 34 cases representing the other most commonly recognized WHO subtypes of B-ALL, determined based on cytogenetic studies performed at the time of diagnosis. No cases of B-ALL with t(5;14) and B-ALL with iAMP21 was represented. Our cohort included 23 pediatric cases and 24 adult cases and the patients ranged from 1 to 82 years old at the time of initial diagnosis. A total of 8 normal bone marrow cases (negative staging bone marrow biopsies for diffuse large B cell lymphoma, neuroblastoma or classic Hodgkin lymphoma) were used as controls. IgJ IHC was performed on B-plus fixed paraffin embedded bone marrow biopsy specimens using a commercially available and validated anti-IgJ monoclonal antibody (clone OTI3B3). Staining of bone marrow samples was performed at 2 different dilutions; tonsil secondary follicles and neoplastic cells from NLPHL were used as technical controls. Cellular staining in the lymphoblasts was scored in a blinded manner by a board certified hematopathologist and a pathologist in training as diffusely positive, partially positive, or negative. Results Cellular staining was distinguishable from background staining due to circulating immunoglobulins and there was almost perfect inter-observer concordance in identifying positive and negative cases (agreement of 98%, kappa test 0.94). All normal bone marrow controls cases were negative for IgJ cellular staining. A total of 11/46 (23%) B-ALL cases demonstrated partial or diffuse cellular staining for IgJ in the lymphoblasts. This included 4/5 (80%) Ph-like cases, 5/7 (71%) Ph+ cases, 1/3 MLL rearranged cases and 1/6 ETV6-RUNX1. All TCF3-PBX1 (0/4), hyperdiploid B-ALL (0/10), hypodiploid B-ALL (0/2), and B-ALL, NOS cases (0/9) were negative for IgJ. Diffuse IgJ staining was restricted to Ph-like (2/4) or Ph+ (2/5) B-ALL subtypes; the positive MLL rearranged and ETV6-RUNX1 B-ALL cases only showed weak partial staining. IgJ protein was significantly expressed in Ph+/Ph-like B-ALL (p&lt;0.0001) and in our cohort, detected these cases with a 75% sensitivity, 95% specificity, 82% positive predictive value and 92% negative predictive value. Conclusion We conclude that IgJ protein expression occurs in a subset of B-ALL, predominantly restricted to Ph+ and Ph-like cases. Although, these findings will need to be validated in larger studies, our results suggest that IgJ IHC, in concert with routine standard cytogenetics studies may be a rapid and cost effective method in identifying Ph-like B-ALL. Disclosures No relevant conflicts of interest to declare.

Imaging Mass Cytometry Reveals the Spatial Architecture of Myelodysplastic Syndromes and Secondary Acute Myeloid Leukemias

Histologic review of bone marrow trephine biopsies is a central component of the diagnostic and treatment response evaluation of hematologic malignancies. Well-validated antibody reagents are routinely used for immunohistochemistry of these samples to provide additional insight into abnormal antigen expression. However, current immunohistochemistry staining protocols are typically limited to only one or two markers simultaneously. Dysplastic changes in cellular morphology and dyssynchronous expression of lineage markers are common features of myelodysplastic syndromes, myeloproliferative neoplasms and secondary acute leukemias. We have integrated the use of multiple diagnostic validated antibody clones with additional antibodies for hematologic lineages and structural proteins to create a 30-marker panel for imaging mass cytometry (IMC). Antibodies included in this panel identify myeloid, lymphoid, erythroid, macrophage, vascular, megakaryocyte and stromal markers as well as markers of cellular proliferation and apoptosis. Through conjugation to elemental metal tags, the entire panel is stained simultaneously on the tissue sample, then acquired by time-of-flight mass spectrometry on a Hyperion instrument (Fluidigm). Antibody staining concentrations and antigen retrieval conditions were optimized for formalin-fixed paraffin-embedded (FFPE) bone marrow to obtain consistent staining for all markers on the panel. Redundant markers for cell populations were selected to provide further internal validation of the observed staining patterns. After data acquisition, cell segmentation algorithms using CellProfiler and ilastik were applied to quantify marker expression in single cells and Phenograph in HistoCAT was used for cell population clustering. Cluster identities for all cells are associated with the original image location in order to plot the spatial arrangement of populations. Using this highly multiplexed panel, we have imaged sets of bone marrow specimens from patients with normal bone marrow morphology and those with myeloid malignancies. We initially confirmed the staining patterns expected for each antibody patterns of co-expression of lineage markers in normal bone marrow samples. We then extended this panel to examine biopsies from patients with myelodysplastic syndrome, myelofibrosis, and secondary acute myeloid leukemia. We found a clear population of CD71+ CD235a+ erythroid cells with strong expression of the proliferative marker Ki67 located within erythroid islands in normal bone marrow samples and MDS. Cell markers of apoptosis and DNA damage are scattered at low frequency throughout the bone marrow in samples with normal bone marrow morphology, but increased clusters of the DNA damage marker phospho-H2AX are observed in selected cases of myelodysplastic syndromes. Overall, this IMC imaging approach is able to extend the current clinical immunostaining for myeloid malignancies by identifying all major bone marrow cell populations. Through highly multiplexed analysis of bone marrow cell populations, the spatial architecture of cell populations and stromal structures can be elucidated, including erythroid islands, lymphoid aggregates and changes in vascular structures with increasing severity of myelofibrosis. In ongoing studies, the development of these imaging techniques for analysis of archived FFPE bone marrow samples is being applied to translational research on hematologic diseases. Disclosures Oh: Kartos Therapeutics: Consultancy; Disc Medicine: Consultancy; PharmaEssentia: Consultancy; Constellation: Consultancy; CTI Biopharma: Consultancy; Celgene/Bristol Myers Squibb: Consultancy; Blueprint Medicines: Consultancy; Novartis: Consultancy; Gilead Sciences: Consultancy; Incyte Corporation: Consultancy.