Infant ALL (iALL) is initiated in utero, most often by rearrangement of the KMT2A gene (KMT2Ar). It carries a very poor prognosis despite a lack of additional oncogenic driver mutations common in childhood ALL. Here, we aimed to identify specific properties of human fetal hematopoietic stem/progenitor cells (HSPC) that promote leukemic transformation in KMT2Ar iALL using molecular, functional and in vivo assays. Through comparison of human fetal HSPC with adult HSPC transcriptomes we derived a fetal-specific gene signature and identified the fetal oncogene LIN28B and its downstream effectors among the top hits. These genes were also expressed in iALL. Functional assays revealed that LIN28B was essential in human fetal liver (FL) CD34+ cells to maintain proliferation and stem-like properties, and support B- and NK-lymphopoiesis. To interrogate the role of LIN28B in iALL, we utilized a human FL-derived CRISPR-Cas9 KMT2A::AFF1 model. In this CRISPRKMT2A::AFF1 model, human FL CD34+ cells fail to transform upon induction of KMT2A::AFF1 translocation in the absence of LIN28B. Furthermore, LIN28B-expressing CRISPRKMT2A::AFF1 leukemias were more proliferative in vitro and in vivo, with this advantage being lost upon LIN28B knockdown. Mechanistic studies showed that LIN28B acts by stabilizing key early B-lymphoid genes, epigenetic regulators, and cell cycle and anti-apoptotic genes. Thus, LIN28B has an essential role in normal human fetal B-lymphopoiesis, and is necessary for the initiation of KMT2A::AFF1 iALL in human fetal cells in the absence of co-operating mutations. LIN28B activity may help explain why KMT2A::AFF1 leukemias are so aggressive, making it a potential target in LIN28B-expressing leukemias.

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.

Genetic alterations alone cannot account for the diverse phenotypes of cancer cells. Even cancers with the same driver mutation show significant transcriptional heterogeneity and varied responses to therapy. However, the mechanisms underpinning this heterogeneity remain under-explored. Here, we find that novel enhancer usage is a common feature in acute lymphoblastic leukemia (ALL). In particular, KMT2A::AFF1 ALL, an aggressive leukemia with a poor prognosis and a low mutational burden, exhibits substantial transcriptional heterogeneity between individuals. Using single cell multiome analysis and extensive chromatin profiling, we reveal that much transcriptional heterogeneity in KMT2A::AFF1 ALL is driven by novel enhancer usage. By generating high resolution Micro Capture-C data in primary patient samples, we identify patient-specific enhancer activity at key oncogenes such as MEIS1 and RUNX2, driving high levels of expression of both oncogenes in a patient-specific manner. Overall, our data show that enhancer heterogeneity is highly prevalent in KMT2A::AFF1 ALL and may be a mechanism that drives transcriptional heterogeneity in cancer more generally.

Although genetic alterations drive carcinogenesis (PMID: 29439951), they alone cannot account for the diverse phenotypes of cancer cells. Even cancers with the same driver mutation show significant transcriptional heterogeneity and varied responses to therapy (PMID: 32807900). However, the mechanisms underpinning this heterogeneity remain under-explored. Aberrant enhancer activity is a hallmark of many cancers, including KMT2A::AFF1 acute lymphoblastic leukemia (ALL; PMID: 37626123), an aggressive leukemia subtype with a poor prognosis (PMID: 32376390) and a nearly mutationally silent genetic landscape (PMID: 25730765). Despite the low mutational burden, KMT2A-rearranged leukemias exhibit substantial heterogeneity between individuals, providing an ideal model to study transcriptional heterogeneity without the confounding effect of numerous cooperating mutations. To validate the extent of transcriptional heterogeneity we first compared two patient-derived KMT2A::AFF1 cell lines (RS4;11 and SEM), identifying 2,361 differentially expressed genes via RNA sequencing. Next, we compared enhancer activity using Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), identifying 3,342 enhancers with significantly altered activity in these KMT2A::AFF1 ALL cell lines. CRISPR-Cas9-based deletion of representative enhancers confirmed cell line-specific activity on their predicted target genes. Using supervised machine learning, we developed a predictive model that identified the KMT2A-complex as a key factor in enhancer activity differences between cell lines. Our previous work showed that KMT2A::AFF1 binds enhancers and regulates them by recruiting a complex that facilitates transcriptional elongation (PMID: 37626123). In line with this, loss of KMT2A::AFF1 or components of the transcription elongation complex significantly diminishes enhancer-promoter interactions and features of active enhancers, implying that it is essential for maintaining enhancer activity (PMID: 37626123). To confirm differential enhancer usage in patients, we used single-cell multiomics (RNA+ATAC) on KMT2A::AFF1 patient blasts, identifying substantial transcriptional heterogeneity and 3,162 differentially active putative enhancers. As ATAC-seq identifies genomic boundary elements in addition to enhancers and promoters, we performed detailed epigenetic profiling of nine KMT2A::AFF1 patient samples (four infants and five children) with a low-cell number optimized chromatin-immunoprecipitation protocol. Employing a deep-learning-based strategy, we reduced intrinsic sample-to-sample noise and dramatically improved the stringency of differential enhancer identification. This approach revealed 2,647 differentially active enhancers, with 61 displaying activity unique to a single patient. We examined whether any unique enhancers were located near prognostically relevant genes and identified putative patient-specific enhancers near MEIS1 and RUNX2. Single-cell expression data revealed elevated MEIS1 and RUNX2 expression in the patient with these unique enhancers. Using Micro-Capture-C (MCC), we confirmed that these enhancers contact the MEIS1 and RUNX2 promoters, implicating these patient specific enhancers in the overexpression of these genes. Overall, our data suggests that enhancer heterogeneity is highly prevalent in KMT2A::AFF1 ALL and appears to be driven by differential KMT2A-complex binding. We identify differential enhancer activity at key oncogenes such as MEIS1 and RUNX2. Analysis of published microarray data shows that overexpression of either gene is associated with significantly reduced overall survival (PMIDs: 20699438, 19880498, 17312329). Taken together, this indicates that enhancer heterogeneity may significantly contribute to the phenotypic diversity observed between patients in ALL.

Although 90% of children with acute lymphoblastic leukemia (ALL) are now cured, the prognosis for infant-ALL remains dismal. Infant-ALL is usually caused by a single genetic hit that arises in utero: an MLL/KMT2A gene rearrangement (MLL-r). This is sufficient to induce a uniquely aggressive and treatment-refractory leukemia compared to older children. The reasons for disparate outcomes in patients of different ages with identical driver mutations are unknown. Using the most common MLL-r in infant-ALL, MLL-AF4, as a disease model, we show that fetal-specific gene expression programs are maintained in MLL-AF4 infant-ALL but not in MLL-AF4 childhood-ALL. We use CRISPR-Cas9 gene editing of primary human fetal liver hematopoietic cells to produce a t(4;11)/MLL-AF4 translocation, which replicates the clinical features of infant-ALL and drives infant-ALL-specific and fetal-specific gene expression programs. These data support the hypothesis that fetal-specific gene expression programs cooperate with MLL-AF4 to initiate and maintain the distinct biology of infant-ALL.

Down syndrome is associated with genome-wide perturbation of gene expression, which may be mediated by epigenetic changes. We perform an epigenome-wide association study on neonatal bloodspots comparing 196 newborns with Down syndrome and 439 newborns without Down syndrome, adjusting for cell-type heterogeneity, which identifies 652 epigenome-wide significant CpGs ( P < 7.67 × 10 −8 ) and 1,052 differentially methylated regions. Differential methylation at promoter/enhancer regions correlates with gene expression changes in Down syndrome versus non-Down syndrome fetal liver hematopoietic stem/progenitor cells ( P < 0.0001). The top two differentially methylated regions overlap RUNX1 and FLI1 , both important regulators of megakaryopoiesis and hematopoietic development, with significant hypermethylation at promoter regions of these two genes. Excluding Down syndrome newborns harboring preleukemic GATA1 mutations ( N = 30), identified by targeted sequencing, has minimal impact on the epigenome-wide association study results. Down syndrome has profound, genome-wide effects on DNA methylation in hematopoietic cells in early life, which may contribute to the high frequency of hematological problems, including leukemia, in children with Down syndrome.

MLL gene rearrangements (MLLr) are a common cause of aggressive, incurable acute lymphoblastic leukemias (ALL) in infants and children, most of which originate in utero. The most common MLLr produces an MLL-AF4 fusion protein. MLL-AF4 promotes leukemogenesis by activating key target genes, mainly through recruitment of DOT1L and increased histone H3 lysine-79 methylation (H3K79me2/3). One key MLL-AF4 target gene is PROM1, which encodes CD133 (Prominin-1). CD133 is a pentaspan transmembrane glycoprotein that represents a potential pan-cancer target as it is found on multiple cancer stem cells. Here we demonstrate that aberrant PROM1/CD133 expression is essential for leukemic cell growth, mediated by direct binding of MLL-AF4. Activation is controlled by an intragenic H3K79me2/3 enhancer element (KEE) leading to increased enhancer-promoter interactions between PROM1 and the nearby gene TAPT1. This dual locus regulation is reflected in a strong correlation of expression in leukemia. We find that in PROM1/CD133 non-expressing cells, the PROM1 locus is repressed by polycomb repressive complex 2 (PRC2) binding, associated with reduced expression of TAPT1, partially due to loss of interactions with the PROM1 locus. Together, these results provide the first detailed analysis of PROM1/CD133 regulation that explains CD133 expression in MLLr ALL.

ABSTRACT Although 90% of children with acute lymphoblastic leukemia (ALL) are now cured 1 , the prognosis of infant-ALL (diagnosis within the first year of life) remains dismal 2 . Infant-ALL is usually caused by a single genetic hit that arises in utero : rearrangement of the MLL/KMT2A gene ( MLL-r ). This is sufficient to give rise to a uniquely aggressive and treatment-refractory leukemia compared to older children with the same MLL-r 3–5 . The reasons for disparate outcomes in patients of different ages with identical driver mutations are unknown. This paper addresses the hypothesis that fetal-specific gene expression programs co-operate with MLL-AF4 to initiate and maintain infant-ALL. Using direct comparison of fetal and adult HSC and progenitor transcriptomes we identify fetal-specific gene expression programs in primary human cells. We show that MLL-AF4 -driven infant-ALL, but not MLL-AF4 childhood-ALL, displays expression of fetal-specific genes. In a direct test of this observation, we find that CRISPR-Cas9 gene editing of primary human fetal liver cells to produce a t(4;11)/ MLL-AF4 translocation replicates the clinical features of infant-ALL and drives infant-ALL-specific and fetal-specific gene expression programs. These data strongly support the hypothesis that fetal-specific gene expression programs co-operate with MLL-AF4 to initiate and maintain the distinct biology of infant-ALL.

The Prestwick library was screened for antibacterial activity or 'antibiotic resistance breaker" (ARB) potential against four species of Gram-negative pathogens. Discounting known antibacterials, the screen identified very few ARB hits, which were strain/drug specific. These ARB hits included antimetabolites (zidovudine, floxuridine, didanosine, and gemcitabine), anthracyclines (daunorubicin, mitoxantrone, and epirubicin), and psychoactive drugs (gabapentin, fluspirilene, and oxethazaine). These findings suggest that there are few approved drugs that could be directly repositioned as adjunct antibacterials, and these will need robust testing to validate efficacy.