Contains Supplementary Figures 1-9. Supplementary Figure 1 shows age of diagnosis of children and indel burden and rearrangement burden for tumours from predisposed children versus those with sporadic disease. Supplementary Figure 2 shows genomic evidence for novel predispositions. Supplementary Figure 3 shows global methylation patterns. Supplementary Figure 4 shows differential gene expression. Supplementary Figure 5 shows mutational signatures. Supplementary Figure 6 shows histological subtypes across predispositions and across somatic drivers. Supplementary Figure 7 shows polyclonal versus clonal enrichment of 11p LOH in normal kidneys. Supplementary Figure 8 shows phylogenies in children with multiple neoplasms and either WT1 or TRIM28 predispositions. Supplementary Figure 9 shows phylogenies in children with non WT1/ non TRIM28 predispositions or sporadic disease.

Chimeric antigen receptor (CAR) T cell therapy for acute myeloid leukemia (AML) is constrained by antigen heterogeneity and shared expression with healthy compartments, and there are often challenges in obtaining autologous T cells from heavily pretreated patients. To address these challenges, we developed universal donor-derived, base-edited, anti-CD33 CAR T cells (BE-CAR33) that used precise multiplexed cytidine deamination to simultaneously disrupt the TRAC, CD52, and CD7 loci to prevent graft-versus-host disease and evade immunotherapy effects. An open-label, nonrandomized, single-center phase 1 study (ISRCTN14430213) evaluated the safety, feasibility, and activity of BE-CAR33 cell therapy ahead of allogeneic stem cell transplantation (allo-SCT) for patients with AML. Eligible participants were aged less than 16 years with relapsed/refractory AML. Five patients were screened, and three were enrolled; one additional adult received BE-CAR33 through compassionate access. Participants received fludarabine, cyclophosphamide, and alemtuzumab followed by 1.2 to 1.8 × 106 BE-CAR33 cells per kilogram. Treatment-emergent adverse events included cytokine release syndrome (grade ≤2), neurotoxicity (grade 3), cytopenias (grade 4), and transient rashes. Two patients demonstrated reduced minimal residual disease and proceeded to allo-SCT. Serial flow cytometry, chimerism quantification, and vector copy number analyses tracked BE-CAR33 T cells until elimination during transplant. Differentially expressed genes included editing signatures and switched from manufacturing-related toward postexpansion effector and exhaustion profiles. Although primary end points were not met, this first-in-human study demonstrated the feasibility of an "off-the-shelf" base-edited CAR T cell approach and informs future multiantigen strategies against AML.

Outcomes for pediatric acute myeloid leukemia (AML) have improved significantly in recent years. However, relapsed and refractory disease remains a significant problem. The chemotherapy burden experienced by these patients makes the translational development of non-genotoxic experimental therapies attractive. We previously reported that the anti-helminth drug mebendazole induces degradation of the transcription factor MYB and has potent anti-AML activity. In the present study, we use CRISPR drop-out screening to identify genes encoding the proapoptotic regulators BAK and NOXA as hits conferring resistance to mebendazole activity in AML cells. Conversely, targeting MCL1 with a BH3-mimetic significantly enhanced the anti-AML activity of mebendazole in both AML cell lines in vitro and pediatric patient-derived xenograft (PDX) AML cells ex vivo. Treatment of mice transplanted with THP-1 AML cells or aggressive infant PDX AML cells with this drug combination significantly impaired disease progression in vivo. Our data indicate that mebendazole-induced MYB degradation in combination with MCL1 targeting is a novel non-genotoxic therapeutic strategy for pediatric AML.

ABSTRACT:Current therapies, including autologous chimeric antigen receptor (CAR) T-cell immunotherapy, fail to cure half of infants with KMT2A-rearranged acute lymphoblastic leukemia (KMT2Ar-ALL), a disease characterized by frequent central nervous system involvement, poor treatment response, early relapse, and lineage switching. More effective treatment strategies, including the availability of off-the-shelf immunotherapies, is particularly relevant in infants. PROM1/CD133 is a direct target of KMT2A-fusion oncoproteins and is expressed on leukemic cells. Allogeneic invariant natural killer T (iNKT) cells, "innately" more powerful effectors than T cells, can be deployed off-the-shelf without risk of acute graft-versus-host disease. Here, we equip iNKT cells with CD19- and/or CD133-targeting CARs, and investigate their antileukemia activity against KMT2Ar-ALL in relevant in vitro and in vivo models. Compared with monospecific counterparts and dual, bispecific CAR T cells, bispecific CD19-CD133 CAR-iNKT cells have a more potent antileukemia activity, effectively targeting both CAR antigen-high and -low leukemia. Bispecific CAR-iNKT cells eradicate medullary and, notably, leptomeningeal leukemia, and induce sustained remissions without discernible hematologic toxicity. Mechanistically, the more potent antileukemia effect of CAR-iNKT cells over CAR T cells is mediated by a pronounced CAR-dependent and CAR antigen-dependent upregulation of the innate activating receptor NKG2D on CAR-iNKT cells, and its engagement by its corresponding ligands on KMT2Ar-ALL cells. This ensures effective leukemia targeting even with downregulation of CD133 or CD19. Thus, by engaging with 2 different types of leukemia-associated antigens, that is, CAR antigens and NKG2D ligands, CAR-iNKT cells provide a powerful platform for the treatment of KMT2Ar-ALL. This approach can be readily adapted for other high-risk malignancies, including those with otherwise difficult to target leptomeningeal involvement.

Contains Supplementary Tables 1-8. Supplementary Table 1 shows characteristics of discovery cohort. Supplementary Table 2 shows genes included in predisposition analysis. Supplementary Table 3 shows evidence underlying novel predispositions. Supplementary Table 4 shows variants that would be detectable by current clinical assays. Supplementary Table 5 shows substitutions and indels. Supplementary Table 6 shows rearrangements. Supplementary Table 7 shows 10,000 most variable probes. Supplementary Table 8 shows differentially expressed genes.

Children with Down syndrome have a 150-fold increased risk of developing myeloid leukaemia (ML-DS). Unusually for a childhood leukaemia, ML-DS arises from a preleukaemic state, termed transient abnormal myelopoiesis (TAM), via a conserved sequence of mutations. Here, we examine the relationship between the genetic and transcriptional evolution of ML-DS from natural variation; a rich collection of primary patient samples and foetal tissues with a range of constitutional karyotypes. We distil transcriptional consequences of each genetic step in ML-DS evolution, utilising single-cell mRNA sequencing, complemented by phylogenetic analyses in progressive disease. We find that transcriptional changes induced by the TAM-defining GATA1 mutations are retained in, and account for most of the ML-DS transcriptome. The GATA1 transcriptome pervades all stages of ML-DS, including progressive disease that had undergone genetic evolution. Our approach delineates the transcriptional evolution of ML-DS and provides an analytical blueprint for distiling consequences of mutations within their pathophysiological context.

Current therapies, including autologous CAR-T immunotherapy, fail to cure half of infants with KMT2A-rearranged acute lymphoblastic leukemia (KMT2Ar-ALL). Here we deploy allogeneic iNKT cells, innately more powerful effectors than T cells, and equip them with CD19- and/or CD133-targeting CARs. Compared to mono-specific counterparts and bi-specific CAR-T, CD19-CD133 bi-specific CAR-iNKT have more potent anti-leukemia activity, they effectively target CAR antigen-low leukemia, eradicate medullary and leptomeningeal leukemia and induce sustained remissions without discernible hematologic toxicity. Mechanistically, dynamic CAR- and CAR antigen-dependent upregulation of the activating innate receptor NKG2D and its engagement by corresponding ligands on KMT2Ar-ALL cells lead to more potent anti-leukemia effect of CAR-iNKT over CAR-T cells, including against CAR antigen-negative leukemia. Thus, by engaging with two different types of leukemia-associated targets, CAR-iNKT provide a powerful platform for the treatment of KMT2Ar-ALL. This approach can be readily adapted for other high-risk malignancies, including those with otherwise difficult to target leptomeningeal involvement. ### Competing Interest Statement AK, TM, AR, HR, NE, BL, CH and R.J.M.B are co-authors of a patent based on the work presented here. AK chairs the scientific advisory board of and holds share options in Arovella Therapeutics. TAM is a shareholder in and consultant for Dark Blue Therapeutics. R.J.M.B.-R. is a co-founder and consultant for Alchemab Therapeutics Ltd, and co-founder of Theraimmune.

Refractory cancers may arise either through the acquisition of resistance mechanisms or represent distinct disease states. The origin of childhood T-cell acute lymphoblastic leukaemia (T-ALL) that does not respond to initial treatment, i.e. refractory disease, is unknown. Refractory T-ALL carries a poor prognosis and cannot be predicted at diagnosis. Here, we perform single cell mRNA sequencing of T-ALL from 58 children (84 samples) who did, or did not respond to initial treatment. We identify a transcriptionally distinctive blast population, exhibiting features of innate-like lymphocytes, as the major source of refractory disease. Evidence of such blasts at diagnosis heralds refractory disease across independent datasets and is associated with survival in a large, contemporary trial cohort. Our findings portray refractory T-ALL as a distinct disease with the potential for immediate clinical utility.

Approximately 10% of children with cancer harbor a mutation in a predisposition gene. In children with the kidney cancer Wilms tumor, the prevalence is as high as 30%. Certain predispositions are associated with defined histological and clinical features, suggesting differences in tumorigenesis. To investigate this, we assembled a cohort of 137 children with Wilms tumor, of whom 71 had a pathogenic germline or mosaic variant. We examined 237 neoplasms (including two secondary leukemias), utilizing whole-genome sequencing, RNA sequencing, and genome-wide methylation, validating our findings in an independent cohort. Tumor development differed in children harboring a predisposition, depending on the variant gene and its developmental timing. Differences pervaded the repertoire of driver events, including high-risk mutations, the clonal architecture of normal kidneys, and the relatedness of neoplasms from the same individual. Our findings indicate that predisposition may preordain Wilms tumorigenesis, suggesting a variant-specific approach to managing children merits consideration.Significance: Tumors that arise in children with a cancer predisposition may develop through the same mutational pathways as sporadic tumors. We examined this question in the childhood kidney cancer, Wilms tumor. We found that certain predispositions dictate the genetic development of tumors, with clinical implications for these children.See related commentary by Brzezinski and Malkin, p. 258

Infant acute lymphoblastic leukemia (ALL) is an aggressive malignancy that has historically been associated with a very poor prognosis. Despite large co-operative international trials and incremental increases in intensity of therapy, there has been no significant improvement in outcome over the last 3 decades. Using representative cases, we highlight the key differences between KMT2A-rearranged and KMT2A-germline infant ALL, and how advances in molecular diagnostics are unpicking KMT2A-germline genetics and guiding treatment reduction. We focus on KM2TA-rearranged infant B-cell ALL where the last few years have seen the emergence of novel therapies which both are more effective and less toxic than conventional chemotherapy. Of these, there is promising early data on the efficacy and tolerability of the bi-specific T-cell engager monoclonal antibody, blinatumomab, as well as the use of autologous and allogeneic chimeric antigen receptor T-cell therapy. We discuss how we can improve risk stratification and incorporate these new agents to replace the most toxic elements of currently deployed intensive chemotherapy schedules with their associated unacceptable toxicity.

Acute lymphoblastic leukaemia (ALL) is the most common paediatric malignancy. While extramedullary manifestations can occur, renal infiltration is rare. We report the case of a previously healthy one-year-old girl who presented with a two-week history of intermittent fever, lethargy, and progressive abdominal distension. On examination, she was lethargic, with bilateral palpable abdominal masses. Laboratory investigations revealed leukocytosis, bicytopenia, and abnormal urinalysis. A peripheral blood film and flow cytometry confirmed B-cell ALL. Ultrasonography demonstrated bilateral renal enlargement, suggestive of leukemic infiltration. Despite massive nephromegaly, she maintained normal renal function, fluid balance, blood pressure, and urine output. Her nephromegaly resolved clinically within four weeks of initiation of multi-agent chemotherapy. Renal involvement in ALL is an uncommon extramedullary manifestation, often asymptomatic and incidentally discovered on imaging. The majority of reported cases show that leukemic infiltration of the kidneys typically does not impair renal function. Nephromegaly in ALL has no established prognostic significance, with most cases resolving following chemotherapy initiation. This case highlights the importance of considering ALL in the differential diagnosis of paediatric patients presenting with unexplained nephromegaly and/or abdominal masses. Although renal dysfunction in this context is rare, close monitoring and careful management to prevent tumor lysis syndrome are of paramount importance to optimise outcomes.

Infant acute lymphoblastic leukemia (iALL) is an aggressive disease that remains a major clinical challenge.1-3 In 70% to 80% of iALL, translocations of KMT2A gene, most commonly with AFF1 [t(4;11) (q21;q23)], produce an oncogenic fusion protein which recruits a large protein complex, "rewriting" epigenetic marks to alter expression of target genes.4-6 Understanding the KMT2A fusion protein complex has been vital for identifying targets such as menin for novel therapies.7 Investigation of genes regulated by KMT2A::AFF1 also remains an important goal. Previously, we showed that one of the most profoundly dysregulated genes in KMT2A::AFF1 leukemia was PROM1, which encodes the cell surface glycoprotein CD133.8 Proliferation of KMT2A::AFF1 ALL cell lines was highly dependent on CD133 expression and PROM1/CD133 was expressed at significantly higher levels in KMT2Ar ALL than in KMT2A germ line ALL.8-10 CD133 has been identified as a marker of stem cells in many cancers11,12 including leukemia,13,14 and is also expressed on normal hematopoietic stem and progenitor cells.15 Expression of CD133 in samples from patients with KMT2A::AFF1 ALL, is often heterogenous and the mechanisms by which CD133 contributes to leukemia biology are unclear.16 Here we investigate the function of CD133 in KMT2A::AFF1 iALL using a primary human fetal liver-derived model of KMT2Ar leukemia17 that recapitulates the pattern of CD133 expression observed in patient samples. We found that CD133 marks an aggressive population of blasts with a stem cell-like signature in KMT2Ar ALL. This provides a rationale for targeting CD133 by pharmacological or immunotherapy-based approaches alongside other treatment modalities. Single guide RNA (Synthego) triplets targeting the PROM1 start codon were used for PROM1 knockout (PROM1 KO) (supplemental Table 1). Cells were electroporated with Cas9-only or Cas9-single guide RNA complexes using a Neon Transfection System (Thermo Fisher) at 1600 V, 10 milliseconds, 3 pulses and recovered overnight in SFEM II (Stemline) supplemented with 10% fetal bovine serum (Invitrogen), 10 nM interleukin-3 and 5 nM interleukin-7 (Peprotech). For CRISPRKMT2A::AFF1 blast coculture, MS5 stromal layers were prepared as described previ-ously.17 About 2000 CRISPRKMT2A::AFF1 blasts were seeded/well in StemSpan SFEM II (serum free medium for culture and expansion of hematopoietic cells) supplemented as above and incubated at 37 degrees C per 5% CO2 with twice-weekly half-volume medium changes. Flow-cytometric readouts were performed weekly with replating onto fresh stromal layers (supplemental Table 2).

Outcomes with CAR T-cells for acute myeloid leukaemia (AML) have been disappointing to date. Targeting of leukaemic stem cells (LSC) is crucial to prevent relapse, but their heterogeneous antigen expression has precluded the discovery of an ideal CAR target. Moreover, existing targets (e.g. CD33, CD123, CLL1) are also expressed on normal haematopoietic stem and progenitor cells (HSPC) -requiring consolidative transplant- and paediatric AML (pAML) LSC knowledge is primarily derived from adults. We generated a single cell pAML dataset, unique in that it is highly enriched for LSC and characterises both their transcriptome and surface proteome (CITEseq). With this high-resolution dataset, we develop a framework for LSC characterisation and identification of targets specifically tailored to pAML LSC. The dataset comprises >210 000 high-quality single cells from 15 pAML patients (15 diagnostic, 2 serial post-induction and 7 serial relapse samples) and 5 healthy controls (3 bone marrow and 2 cord blood). Median age was 4 years (range 0.2-18) and cytogenetic heterogeneity was well represented: 4 KMT2Ar, 2 inv(16), 2 NUP98::NSD1, 1 t(8;21), 1 CBFA2::GLIS2, 1 t(6;9) and 1 t(7;12)+19 (cytogenetic information was unavailable for 3 patients). Cells were stained with a custom panel of 81 CITEseq antibodies against potential LSC markers, sorted and enriched for CD34 (median 70%). Transcriptome and surface proteome libraries were generated with 10X Genomics. We first differentiated malignant from healthy cells using an occupancy score, presence of copy number variations and a 7-gene pAML blast signature (PMID: 37798266) and validated this annotation with a machine learning classifier (scANVI). Cell-types/states were obtained by mapping our single cell transcriptomes to published datasets. Our integrated analysis identified a cell cluster highly enriched in functionally derived LSC gene expression signatures, with contributions from all AML samples (median 4.7% LSC, IQR 1.3-6.7). These cells express a unique gene program (e.g., higher MSI2, RUNX1 and IGF2BP2 expression) that is distinct from adult LSC and adult healthy HSPC signatures, but is recapitulated in cord blood, potentially reflecting their cell of origin. This may allow preferential targeting of pAML LSC over mature HSPC. By analysing the surface proteome (custom CITEseq panel) and leveraging transcriptomes to discover surface-protein-coding genes, we identified targets overexpressed in LSC vs healthy HSPC, at the transcript (181) and protein (37) levels. Among these is CD84, a recently proposed pAML CAR target (Pigazzi et al., ASH 2022), restricted to haematopoietic tissue. High expression in LSC (56% across all patients, IQR 37-77) and blasts (83%, IQR 56-92) and low levels in healthy HSPC (21%, IQR 18-23, p<2.2-16) and lymphocytes are in contrast with CD33, CD123 and CLL1, which -while expressed in 51-65% of LSC- are expressed by 47-82% HSPC (p<0.05). These data suggest that CD84 may be ideal to target AML LSC whilst preserving normal HSPC, overriding the need for consolidative transplant. Further, other LSC targets are enriched in distinct pAML groups driven by cytogenetics and cellular hierarchies (PMID: 35618837), paving the way for rationally designing combinatorial strategies that enhance efficacy and limit toxicity. Here we present a unique dataset to profile vulnerabilities in pAML LSC and provide a framework to identify pAML LSC-specific targets and tailor CAR target combinations, with CD84 emerging as a potential near-universal LSC marker. We are currently validating and expanding our findings in silico by applying our paediatric LSC signature to other AML datasets and using predictive algorithms to infer protein levels from single cell transcriptomes lacking protein information. A systematic analysis to elicit optimal target combinations, as well as functional validation of candidate and previously undescribed paediatric LSC markers are underway. *MOE and SK contributed equally

CD19-negative relapse is a leading cause of treatment failure after chimeric antigen receptor (CAR) T-cell therapy for acute lymphoblastic leukemia. We investigated a CAR T-cell product targeting CD19 and CD22 generated by lentiviral cotransduction with vectors encoding our previously described fast -off rate CD19 CAR (AUTO1) combined with a novel CD22 CAR capable of effective signaling at low antigen density. Twelve patients with advanced B-cell acute lymphoblastic leukemia were treated (CARPALL [Immunotherapy with CD19/22 CAR Redirected T Cells for High Risk/Relapsed Paediatric CD19+ and/or CD22+ Acute Lymphoblastic Leukaemia] study, NCT02443831), a third of whom had failed prior licensed CAR therapy. Toxicity was similar to that of AUTO1 alone, with no cases of severe cytokine release syndrome. Of 12 patients, 10 (83%) achieved a measurable residual disease (MRD)-negative complete remission at 2 months after infusion. Of 10 responding patients, 5 had emergence of MRD (n = 2) or relapse (n = 3) with CD19- and CD22-expressing disease associated with loss of CAR T-cell persistence. With a median follow-up of 8.7 months, there were no cases of relapse due to antigen -negative escape. Overall survival was 75% (95% confidence interval [CI], 41%-91%) at 6 and 12 months. The 6- and 12-month event-free survival rates were 75% (95% CI, 41%-91%) and 60% (95% CI, 23%-84%), respectively. These data suggest dual targeting with cotransduction may prevent antigen -negative relapse after CAR T-cell therapy.

Chimeric antigen receptor (CAR) T -cell therapy has transformed the landscape of relapsed/refractory B -cell acute lymphoblastic leukemia (ALL) in children and young adults, (1,2) with a 3 -year relapse -free survival of 52% for tisagenlecleucel in the pivotal ELIANA trial. 3 B -cell aplasia (BCA) is an indirect measure of anti-CD19 CAR T -cell presence. Early ( <= 6 months from infusion) loss of BCA (LBCA) was associated with high relapse risk in studies with tisagenlecleucel or other 41BBz anti-CD19 CAR T -cell products. (4-9) However, with different anti-CD19 CAR T cells (eg, CD28-containing brexucabtagene), the long-term persistence of CAR T cells seems not required for durable remission. (10) The optimal therapeutic strategy for patients with early LBCA after tisagenlecleucel is unclear; good outcomes have been achieved with consolidative hematopoietic stem cell transplant (SCT). (3,9) However, SCT is associated with signi fi cant mortality, especially for patients with prior SCT within 12 months,( 11) and with long-term side effects. (12) Indeed, a benefit of consolidative SCT after CAR T -cell therapy has not been demonstrated for patients who have received prior transplant. (13) Moreover, not all patients have a suitable donor, and some are precluded from SCT because of comorbidities. At our center, children who received tisagenlecleucel and presented with early LBCA with a contraindication to SCT were treated with a maintenance chemotherapy regimen for 2 years, with promising early outcomes. (14 )Here, we report on the longer follow-up of a larger cohort of children and young adults from across the United Kingdom receiving maintenance chemotherapy or SCT after early LBCA. We retrospectively collected data of patients treated either with tisagenlecleucel or experimental 41BBz anti-CD19 and anti-CD19/anti-CD22 CART cells (NCT02443831) in the United Kingdom from June 2017 to June 2022. The data cut-off date was 20 March 2023. Data were collected on a health service evaluation basis, on the basis of outcome assessment after CAR T -cell therapy. Consent for data collection was obtained from patients, parents, or legal guardians. Inclusion criteria were early LBCA ( <= 6 months from infusion) without evidence of disease, de fi ned as morphological complete remission (CR) and negative measurable residual disease (MRD) by polymerase chain reaction analysis.