Addressing a Pre-Clinical Pipeline Gap: Development of the Pediatric Acute Myeloid Leukemia Patient-Derived Xenograft Program at Texas Children's Hospital at Baylor College of Medicine.

The survival rate of pediatric acute myeloid leukemia (pAML) is currently around 60%. While survival has slowly increased over the past few decades, the development of novel agents likely to further improve survival for this heterogeneous patient population has been limited by gaps in the pAML pre-clinical pipeline. One of the major hurdles in evaluating new agents for pAML is the lack of pAML patient-derived xenograft (PDX) models. Unlike solid tumors and other types of leukemias, AML is notoriously hard to establish in mouse models, likely due in part to the need for specific human microenvironment elements. Our laboratory at TCH/BCM addressed this gap by establishing a systematic PDX workflow, leveraging advanced immunodeficient hosts and capitalizing on our high volume of pAML patients and close coordination between labs and clinical sections. Patients treated at TCH are offered the chance to participate in specimen banking protocols that allow blood and bone marrow collection as well as the collection of relevant clinical data. All patients who consent and have samples available are trialed for PDX development. In addition, samples from the Children's Oncology Group (COG) are also trialed for PDX generation. Serially transplanting PDX models are validated using short tandem repeat (STR) and characterized using both targeted DNA/RNA next generation sequencing and RNAseq. As of March 2023, this systematic approach has resulted in 26 serially transplanting models. Models have been shared with requesting labs to facilitate external pAML pre-clinical studies. Available PDX models can be located through the BCM PDX Portal. We expect our growing PDX resource to make a significant contribution to expediting the testing of promising novel therapeutics for pAML.

ALK Fusion in an Adolescent with Acute Myeloid Leukemia: A Case Report and Review of the Literature.

Activating mutations and fusions of the ALK oncogene have been identified as drivers in a number of malignancies. Crizotinib and subsequent ALK tyrosine kinase inhibitors have improved treatment outcomes for these patients. In this paper, we discuss the case of an adolescent patient with acute myeloid leukemia, who was identified to have an activating ALK fusion, which is a rare finding and has never been reported in cases of AML without monosomy 7. Crizotinib was added to this patient's frontline therapy and was well tolerated. In cases of more common gene alterations, existing data supports the use of targeted agents as post-HSCT maintenance therapy; however, crizotinib was not able to be used post-HSCT for this patient due to the inability to obtain insurance coverage.

Repurposing Atovaquone as a Therapeutic against Acute Myeloid Leukemia (AML): Combination with Conventional Chemotherapy Is Feasible and Well Tolerated.

Survival of pediatric AML remains poor despite maximized myelosuppressive therapy. The pneumocystis jiroveci pneumonia (PJP)-treating medication atovaquone (AQ) suppresses oxidative phosphorylation (OXPHOS) and reduces AML burden in patient-derived xenograft (PDX) mouse models, making it an ideal concomitant AML therapy. Poor palatability and limited product formulations have historically limited routine use of AQ in pediatric AML patients. Patients with de novo AML were enrolled at two hospitals. Daily AQ at established PJP dosing was combined with standard AML therapy, based on the Medical Research Council backbone. AQ compliance, adverse events (AEs), ease of administration score (scale: 1 (very difficult)-5 (very easy)) and blood/marrow pharmacokinetics (PK) were collected during Induction 1. Correlative studies assessed AQ-induced apoptosis and effects on OXPHOS. PDX models were treated with AQ. A total of 26 patients enrolled (ages 7.2 months-19.7 years, median 12 years); 24 were evaluable. A total of 14 (58%) and 19 (79%) evaluable patients achieved plasma concentrations above the known anti-leukemia concentration (>10 µM) by day 11 and at the end of Induction, respectively. Seven (29%) patients achieved adequate concentrations for PJP prophylaxis (>40 µM). Mean ease of administration score was 3.8. Correlative studies with AQ in patient samples demonstrated robust apoptosis, OXPHOS suppression, and prolonged survival in PDX models. Combining AQ with chemotherapy for AML appears feasible and safe in pediatric patients during Induction 1 and shows single-agent anti-leukemic effects in PDX models. AQ appears to be an ideal concomitant AML therapeutic but may require intra-patient dose adjustment to achieve concentrations sufficient for PJP prophylaxis.

IL-10 and TNFα are associated with decreased survival in low-risk pediatric acute myeloid leukemia; a children's oncology group report.

Pediatric acute myeloid leukemia (AML) is a devastating disease with a high risk of relapse. Current risk classification designates patients as high or low risk (LR) based on molecular features and therapy response. However, 30% of LR patients still suffer relapse, indicating a need for improvement in risk stratification. Cytokine levels, such as IL-6 and IL-10, have been shown to be prognostic in adult AML but have not been well studied in children. Previously, we reported elevated IL-6 levels in pediatric AML bone marrow to be associated with inferior prognosis. Here, we expanded our investigation to assess cytokine levels in diagnostic peripheral blood plasma (PBP) of pediatric AML patients and determined correlation with outcome. Diagnostic PBP was obtained from 80 patients with LR AML enrolled on the Children's Oncology Group AAML1031 study and normal PBP from 11 controls. Cytokine levels were measured and correlation with clinical outcome was assessed. IL-6, TNFα, MIP-3a, and IL-1ß were significantly higher in AML patients versus controls when corrected by the Bonferroni method. Furthermore, elevated TNFα and IL-10 were significantly associated with inferior outcomes. Our data demonstrate that in diagnostic PBP of LR pediatric AML patients, certain cytokine levels are elevated as compared to healthy controls and that elevated TNFα and IL-10 are associated with inferior outcomes, supporting the idea that an abnormal inflammatory state may predict poor outcomes. Studies are needed to determine the mechanisms by which these cytokines impact survival, and to further evaluate their use as prognostic biomarkers in pediatric AML.

Comparison of the Transcriptomic Signatures in Pediatric and Adult CML.

Children with chronic myeloid leukemia (CML) tend to present with higher white blood counts and larger spleens than adults with CML, suggesting that the biology of pediatric and adult CML may differ. To investigate whether pediatric and adult CML have unique molecular characteristics, we studied the transcriptomic signature of pediatric and adult CML CD34+ cells and healthy pediatric and adult CD34+ control cells. Using high-throughput RNA sequencing, we found 567 genes (207 up- and 360 downregulated) differentially expressed in pediatric CML CD34+ cells compared to pediatric healthy CD34+ cells. Directly comparing pediatric and adult CML CD34+ cells, 398 genes (258 up- and 140 downregulated), including many in the Rho pathway, were differentially expressed in pediatric CML CD34+ cells. Using RT-qPCR to verify differentially expressed genes, VAV2 and ARHGAP27 were significantly upregulated in adult CML CD34+ cells compared to pediatric CML CD34+ cells. NCF1, CYBB, and S100A8 were upregulated in adult CML CD34+ cells but not in pediatric CML CD34+ cells, compared to healthy controls. In contrast, DLC1 was significantly upregulated in pediatric CML CD34+ cells but not in adult CML CD34+ cells, compared to healthy controls. These results demonstrate unique molecular characteristics of pediatric CML, such as dysregulation of the Rho pathway, which may contribute to clinical differences between pediatric and adult patients.

Introduction to the Special Issue on Pediatric Acute Myeloid Leukemia: Current Management and Future Directions.

This Special Issue brings together an original research report, a fascinating case report, and three timely reviews on a variety of topics related to pediatric leukemia [...].

Modulating TNFα activity allows transgenic IL15-Expressing CLL-1 CAR T cells to safely eliminate acute myeloid leukemia.

BACKGROUND: C-type lectin-like molecule 1 (CLL-1) is highly expressed in acute myeloid leukemia (AML) but is absent in primitive hematopoietic progenitors, making it an attractive target for a chimeric antigen receptor (CAR) T-cell therapy. Here, we optimized our CLL-1 CAR for anti-leukemic activity in mouse xenograft models of aggressive AML. METHODS: First, we optimized the CLL-1 CAR using different spacer, transmembrane and costimulatory sequences. We used a second retroviral vector to coexpress transgenic IL15. We measured the effects of each construct on T cell phenotype and sequential (recursive) co culture assays with tumor cell targets to determine the durability of the anti tumor activity by flow cytometry. We administered CAR T cells to mice engrafted with patient derived xenografts (PDX) and AML cell line and determined anti tumor activity by bioluminescence imaging and weekly bleeding, measured serum cytokines by multiplex analysis. After euthanasia, we examined formalin-fixed/paraffin embedded sections. Unpaired two-tailed Student's t-tests were used and values of p<0.05 were considered significant. Survival was calculated using Mantel-Cox log-rank test. RESULTS: In vitro, CLL-1 CAR T cells with interleukin-15 (IL15) were less terminally differentiated (p<0.0001) and had superior expansion compared with CD28z-CD8 CAR T cells without IL15 (p<0.001). In both AML PDX and AML cell line animal models, CLL-1 CAR T coexpressing transgenic IL15 initially expanded better than CD28z-CD8 CAR T without IL15 (p<0.0001), but produced severe acute toxicity associated with high level production of human tumor necrosis factor α (TNFα), IL15 and IL2. Histopathology showed marked inflammatory changes with tissue damage in lung and liver. This acute toxicity could be managed by two strategies, individually or in combination. The excessive TNF alpha secretion could be blocked with anti-TNF alpha antibody, while excessive T cell expansion could be arrested by activation of an inducible caspase nine safety switch by administration of dimerizing drug. Both strategies successfully prolonged tumor-free survival. CONCLUSION: Combinatorial treatment with a TNFα blocking antibody and subsequent activation of the caspase-9 control switch increased the expansion, survival and antileukemic potency of CLL-1 CAR T-cells expressing transgenic IL15 while avoiding the toxicities associated with excessive cytokine production and long-term accumulation of activated T-cells.

Targeting STAT3 anti-apoptosis pathways with organic and hybrid organic-inorganic inhibitors.

Recurrence and drug resistance are major challenges in the treatment of acute myeloid leukemia (AML) that spur efforts to identify new clinical targets and active agents. STAT3 has emerged as a potential target in resistant AML, but inhibiting STAT3 function has proven challenging. This paper describes synthetic studies and biological assays for a naphthalene sulfonamide inhibitor class of molecules that inhibit G-CSF-induced STAT3 phosphorylation in cellulo and induce apoptosis in AML cells. We describe two different approaches to inhibitor design: first, variation of substituents on the naphthalene sulfonamide core allows improvements in anti-STAT activity and creates a more thorough understanding of anti-STAT SAR. Second, a novel approach involving hybrid sulfonamide-rhodium(ii) conjugates tests our ability to use cooperative organic-inorganic binding for drug development, and to use SAR studies to inform metal conjugate design. Both approaches have produced compounds with improved binding potency. In vivo and in cellulo experiments further demonstrate that these approaches can also lead to improved activity in living cells, and that compound 3aa slows disease progression in a xenograft model of AML.

Pediatric myeloid sarcoma: a single institution clinicopathologic and molecular analysis.

Myeloid sarcoma (MS) is a neoplastic condition composed of immature myeloid cells involving an extramedullary site. We investigated underlying chromosomal and molecular alterations to assess potential molecular markers of prognosis and outcome in this rare pediatric disease. We conducted a retrospective review of clinicopathologic and cytogenetic data from 33 pediatric patients with MS (ages 1 month-18 years) at our institution over a 32 year period (1984-2016). Tissue-based cancer microarray and targeted next-generation sequencing analysis were performed on six cases. The median age at diagnosis was 2.8 years with a male-to-female ratio of 2.6:1. MS is commonly presented with concomitant marrow involvement (n = 12, 36.4%) or as a recurrence of acute myeloid leukemia (AML; n = 14, 42.4%). The skin (n = 18, 54.5%) and soft tissue (n = 9, 27.3%) were the most common sites of involvement. Twenty-one of 25 samples (84.0%) harbored chromosomal aberrations; KMT2A alterations (n = 10, 40.0%) or complex cytogenetics (n = 7, 28.0%) were most frequent. Mutations in RAS, tyrosine kinase, cell signaling, and chromatin remodeling genes were detected. When compared to pediatric patients with AML without extramedullary involvement (EMI), inferior overall survival (OS) was observed (18.8 months vs. 89.3 months, p = .008). Pediatric patients with MS with non-favorable cytogenetics [abnormalities other than t(8;21), inv(16)/t(16;16), or t(15;17)] had a significantly lower OS compared to patients with AML with non-favorable cytogenetics and no extramedullary involvement (8.0 months vs. 28.1 months, p < .001). Pediatric MS is a rare disease with diverse clinical presentations. Non-favorable cytogenetics may be a poor prognostic marker for pediatric patients with MS and molecular diagnostics can assist with risk stratification and identify potentially actionable targets.

Atovaquone is active against AML by upregulating the integrated stress pathway and suppressing oxidative phosphorylation.

Atovaquone, a US Food and Drug Administration-approved antiparasitic drug previously shown to reduce interleukin-6/STAT3 signaling in myeloma cells, is well tolerated, and plasma concentrations of 40 to 80 µM have been achieved with pediatric and adult dosing. We conducted preclinical testing of atovaquone with acute myeloid leukemia (AML) cell lines and pediatric patient samples. Atovaquone induced apoptosis with an EC50 <30 µM for most AML lines and primary pediatric AML specimens. In NSG mice xenografted with luciferase-expressing THP-1 cells and in those receiving a patient-derived xenograft, atovaquone-treated mice demonstrated decreased disease burden and prolonged survival. To gain a better understanding of the mechanism of atovaquone, we performed an integrated analysis of gene expression changes occurring in cancer cell lines after atovaquone exposure. Atovaquone promoted phosphorylation of eIF2α, a key component of the integrated stress response and master regulator of protein translation. Increased levels of phosphorylated eIF2α led to greater abundance of the transcription factor ATF4 and its target genes, including proapoptotic CHOP and CHAC1. Furthermore, atovaquone upregulated REDD1, an ATF4 target gene and negative regulator of the mechanistic target of rapamycin (mTOR), and caused REDD1-mediated inhibition of mTOR activity with similar efficacy as rapamycin. Additionally, atovaquone suppressed the oxygen consumption rate of AML cells, which has specific implications for chemotherapy-resistant AML blasts that rely on oxidative phosphorylation for survival. Our results provide insight into the complex biological effects of atovaquone, highlighting its potential as an anticancer therapy with novel and diverse mechanisms of action, and support further clinical evaluation of atovaquone for pediatric and adult AML.

Management of chronic myeloid leukemia in children and adolescents: Recommendations from the Children's Oncology Group CML Working Group.

Chronic myeloid leukemia (CML) accounts for 2-3% of leukemias in children under 15 and 9% in adolescents aged 15-19. The diagnosis and management of CML in children, adolescents, and young adults have several differences compared to that in adults. This review outlines the diagnosis and management of the underlying disease as well as challenges that can occur when dealing with CML in this patient population.

Drug targeting of NR4A nuclear receptors for treatment of acute myeloid leukemia.

NR4As are AML tumor suppressors that are frequently silenced in human acute myeloid leukemia (AML). Despite their potential as novel targets for therapeutic intervention, mechanisms of NR4A silencing and strategies for their reactivation remain poorly defined. Here we show that NR4A silencing in AML occurs through blockade of transcriptional elongation rather than epigenetic promoter silencing. By intersection of NR4A-regulated gene signatures captured upon acute, exogenous expression of NR4As in human AML cells with in silico chemical genomics screening, we identify several FDA-approved drugs including dihydroergotamine (DHE) that reactivate NR4A expression and regulate NR4A-dependent gene signatures. We show that DHE induces NR4A expression via recruitment of the super elongation complex to enable elongation of NR4A promoter paused RNA polymerase II. Finally, DHE exhibits AML selective NR4A-dependent anti-leukemic activity in cytogenetically distinct human AML cells in vitro and delays AML progression in mice revealing its potential as a novel therapeutic agent in AML.

Distinct signaling events promote resistance to mitoxantrone and etoposide in pediatric AML: a Children's Oncology Group report.

Despite aggressive chemotherapy including mitoxantrone and etoposide, relapse occurs for almost half of children with acute myeloid leukemia (AML). Since both drugs inhibit topoisomerase II and cause DNA double strand breaks, resistance could be achieved by enhanced DNA damage repair (DDR), via homologous recombination (HR) and/or non-homologous end joining (NHEJ). An important source of extrinsic chemoresistance is the bone marrow stroma. We aimed to reveal intrinsic and stroma-induced signaling pathways that contribute to chemoresistance. Sixty diagnostic pediatric AML samples were cultured on or off stromal cells, with or without chemotherapy. We measured apoptosis, DNA damage signaling, and NHEJ/HR pathway activity by FACS analysis of intracellular cleaved PARP, γH2AX, pDNA-PKcs and pATM, respectively. Mitoxantrone strongly increased γH2AX and pDNA-PKcs. Neither chemotherapy drug induced pATM. DNA-PK inhibition alleviated mitoxantrone resistance for AML cells on and off stromal cells. Regarding stroma-induced signaling pathways, ERK1/2 was most consistently activated in primary AML cells by stromal cells. ERK1/2 inactivation partially restored chemosensitivity to AML cells on stromal cells. Additionally, low stroma-induced STAT3 activity and strong stroma-induced mitoxantrone resistance were associated with inferior clinical outcome. Taken together, the NHEJ DDR and ERK1/2 pathways are potential targets for reducing intrinsic and extrinsic chemotherapy resistance in pediatric AML.

Interleukin-6 levels predict event-free survival in pediatric AML and suggest a mechanism of chemotherapy resistance.

The tumor microenvironment can protect cancer cells from conventional anticancer therapies. Thus, targeting these protective mechanisms could eradicate therapy-resistant cancer cells and improve outcomes. Interleukin-6 (IL-6) provides extrinsic protection for several solid tumors and multiple myeloma. In pediatric acute myeloid leukemia (AML), IL-6-induced STAT3 signaling frequently becomes stronger at relapse, and increases in IL-6-induced STAT3 activity are associated with inferior survival after relapse. These findings suggested that the IL-6-induced STAT3 pathway may promote chemotherapy resistance and disease progression. Thus, we investigated the dysregulation of IL-6 levels in the bone marrow niche in pediatric patients with AML and the association between IL-6 levels and outcome. We measured levels of over 40 cytokines and growth factors in plasma from diagnostic bone marrow aspirates of 45 pediatric AML patients and 7 healthy sibling controls. Of the measured cytokines, only IL-6 levels were associated with event-free survival. Importantly, the effect of elevated IL-6 was most striking among children classified as having a low risk of relapse. In these patients, 5-year event-free survival was 82.5% ± 11% for patients with low IL-6 levels at diagnosis (n = 14) compared with 17.3% ± 11% for patients with elevated IL-6 (n = 13, log-rank P = .0003). In vitro, exogenous IL-6 reduced mitoxantrone-induced apoptosis in cell lines and primary pediatric AML samples. These results suggest that IL-6 levels at diagnosis could be used to help identify children at high risk of relapse, particularly those who are otherwise classified as low risk by current algorithms. Moreover, the IL-6 pathway could represent a target for overcoming environment-mediated chemotherapy resistance.

Assessing the intracellular fate of rhodium(ii) complexes.

Rhodium(ii)-fluorophore conjugates have strong rhodium-based fluorescence quenching that can be harnessed to report on a conjugate's cellular uptake and the intracellular decomposition rate. Information gleened from this study allowed the design of an improved STAT3 metalloinhibitor.

A STAT3 decoy lures AML out of hiding.

The transcription factor signal transducer and activator of transcription 3 (STAT3) is perhaps best known for its prosurvival effects in a wide variety of cancers, but for some, including acute myeloid leukemia (AML), its role in immune evasion may be just as important. In this issue of Blood, Zhang et al report the development of an engineered STAT3 decoy oligodeoxynucleotide (dODN) that is stable in serum, is taken up specifically by target cells, and exerts its antileukemia effects largely by restoring the host anti-AML immune response.

Rhodium(II) Proximity-Labeling Identifies a Novel Target Site on STAT3 for Inhibitors with Potent Anti-Leukemia Activity.

Nearly 40 % of children with acute myeloid leukemia (AML) suffer relapse arising from chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). Herein, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML.

Stromal CYR61 Confers Resistance to Mitoxantrone via Spleen Tyrosine Kinase Activation in Human Acute Myeloid Leukaemia.

Approximately 50% of children with acute myeloid leukaemia (AML) relapse, despite aggressive chemotherapy. The bone marrow stromal environment protects leukaemia cells from chemotherapy (i.e., stroma-induced chemoresistance), eventually leading to recurrence. Our goal is to delineate the mechanisms underlying stroma-mediated chemoresistance in AML. We used two human bone marrow stromal cell lines, HS-5 and HS-27A, which are equally effective in protecting AML cells from chemotherapy-induced apoptosis in AML-stromal co-cultures. We found that CYR61 was highly expressed by stromal cells, and was upregulated in AML cells by both stromal cell lines. CYR61 is a secreted matricellular protein and is associated with cell-intrinsic chemoresistance in other malignancies. Here, we show that blocking stromal CYR61 activity, by neutralization or RNAi, increased mitoxantrone-induced apoptosis in AML cells in AML-stromal co-cultures, providing functional evidence for its role in stroma-mediated chemoresistance. Further, we found that spleen tyrosine kinase (SYK) mediates CYR61 signalling. Exposure to stroma increased SYK expression and activation in AML cells, and this increase required CYR61. SYK inhibition reduced stroma-dependent mitoxantrone resistance in the presence of CYR61, but not in its absence. Therefore, SYK is downstream of CYR61 and contributes to CYR61-mediated mitoxantrone resistance. The CYR61-SYK pathway represents a potential target for reducing stroma-induced chemoresistance.