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.

Children with Down syndrome (DS) frequently develop transient abnormal myelopoiesis that can evolve to the myeloid leukemia of DS (ML-DS). TAM spontaneously resolves in most cases but progresses to ML-DS with additional mutations, most commonly in the cohesin complex. However, the mechanisms by which these alterations promote leukemia are unknown. We leveraged the RAD21-mutant CMY cell line and RAD21-corrected CMY isogenic clones, as well as patient data, to investigate the effect of cohesin mutations during leukemia progression. RNA-sequencing revealed that HLA-class II genes were significantly down-regulated with cohesin mutations. Furthermore, HLA-DR was found to be lower in ML-DS relative to TAM, and these decreased levels were associated with increased risk of leukemia progression. Multi-omic analyses revealed that haploinsufficiency of RAD21 altered chromatin accessibility and impaired the occupancy of GATA1s and CIITA, the master regulator of HLA-class II gene expression. Chromatin binding of CIITA was increased with RAD21 correction, providing a mechanism by which restoration of cohesin improves HLA-class II expression. Finally, decreased levels of RAD21 or STAG2 expression in other subtypes of AML also exhibited reduced expression of HLA-class II genes. Thus, cohesin may contribute to leukemia by altering HLA-class II gene expression.

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.

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).

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.

PURPOSE Failure to respond to induction chemotherapy portends a poor outcome in childhood acute lymphoblastic leukemia (ALL) and is more frequent in T-cell ALL (T-ALL) than B-cell ALL. We aimed to address the limited understanding of clinical and genetic factors that influence outcome in a cohort of patients with T-ALL induction failure (IF). METHODS We studied all cases of T-ALL IF on two consecutive multinational randomized trials, UKALL2003 and UKALL2011, to define risk factors, treatment, and outcomes. We performed multiomic profiling to characterize the genomic landscape. RESULTS IF occurred in 10.3% of cases and was significantly associated with increasing age, occurring in 20% of patients age 16 years and older. Five-year overall survival (OS) rates were 52.1% in IF and 90.2% in responsive patients ( P < .001). Despite increased use of nelarabine-based chemotherapy consolidated by hematopoietic stem-cell transplant in UKALL2011, there was no improvement in outcome. Persistent end-of-consolidation molecular residual disease resulted in a significantly worse outcome (5-year OS, 14.3% v 68.5%; HR, 4.10; 95% CI, 1.35 to 12.45; P = .0071). Genomic profiling revealed a heterogeneous picture with 25 different initiating lesions converging on 10 subtype-defining genes. There was a remarkable abundance of TAL1 noncoding lesions, associated with a dismal outcome (5-year OS, 12.5%). Combining TAL1 lesions with mutations in the MYC and RAS pathways produces a genetic stratifier that identifies patients highly likely to fail conventional therapy (5-year OS, 23.1% v 86.4%; HR, 6.84; 95% CI, 2.78 to 16.78; P < .0001) and who should therefore be considered for experimental agents. CONCLUSION The outcome of IF in T-ALL remains poor with current therapy. The lack of a unifying genetic driver suggests alternative approaches, particularly using immunotherapy, are urgently needed.

Background: CD19 negative escape is a major cause of relapse after CD19 CAR T cell therapy for relapsed/refractory (r/r) paediatric Acute Lymphoblastic Leukemia (ALL) and dual targeting of CD19/CD22 may overcome this. We have previously shown that AUTO1, a fast off rate autologous CD19 CAR T cell therapy was highly active in ALL with a favorable safety profile and excellent persistence (Ghorashian et al Nat.Med. 2019). Building on these properties, we developed AUTO1/22 an autologous CAR T cell product co-transduced with two different lentiviral vectors encoding our existing CD19 CAR and a novel CD22CAR designed to recognise targets with low antigen density. We have evaluated the safety and biological efficacy of AUTO1/22 in a Phase I study in children/young adults with r/r ALL (NCT02443831). Methods: Patients with r/r B-ALL age < 25 yrs who were ineligible for/relapsed after Tisagenlecleucel were recruited. Following fludarabine/cyclophosphamide lymphodepletion, patients received 1x106 /kg CAR+ T cells. The presence of CAR T cells in the blood/bone marrow (BM) was assessed by flow cytometry + qPCR and BM MRD was assessed by IgH qPCR + flow cytometry. Primary endpoints were incidence of grade 3-5 toxicity and the proportion of patients achieving MRD negative remission. Results: 12 patients have been treated. The median age was 12 years (range 3-21) and patients had a median of 3 prior lines of therapy (range 2-6). Six of 12 patients had relapsed post allogeneic SCT, 6 had received prior Blinatumomab/Inotuzumab and 4 had relapsed after prior Tisagenlecleucel. Prior to lymphodepletion, 4 patients had >5% BM disease by morphology/flow, 5 had detectable BM MRD and 3 were BM MRD negative. Six patients had extramedullary relapse (of which 2 had non-CNS EM disease). Three had detectable CD19 negative disease at enrolment. One of these was completely CD19 negative and in addition had a 5% CD22 negative population. CAR T cell products had a central memory phenotype with predominance of CD19/22CAR double positive cells (median 54.4%) and balanced populations of CD19 and CD22 single positive cells (13.1% and 11.6% respectively). Cytokine release syndrome (CRS) occurred in 11/12 patients (grade 1 n=5, grade 2 n=6) requiring Tocilizumab in 5 cases, but severe (≥ grade 3) CRS was not seen and no patients required ICU admission for CRS. Grade 1-2 ICANS was observed in 5 patients. One patient had delayed grade 4 leucoencephalopathy (MRI/brain biopsy were more indicative of fludarabine toxicity than CAR T related) and has ongoing neurological recovery. Nine patients had grade 3-4 cytopenia persisting beyond/recurring after day 28, requiring a CD34+ stem cell top up in 1 case. Flow cytometry showed significant initial expansion of all 3 CAR +ve populations early post-infusion but CD19/22CAR double positive cells were lost at later time points. Six patients had circulating CAR T cells by qPCR at last follow up and the median duration of B cell aplasia has not been reached. Ten of 12 patients (83%) achieved MRD negative CR/CRi at one month post-infusion and 2 patients did not respond. Importantly 2/3 patients with CD19-ve disease achieved molecular CR demonstrating the efficacy of our CD22CAR. Of the 10 responding patients, 2 have relapsed with CD19+CD22+ disease, one patient with CD19-ve disease pre-infusion has relapsed with CD19partialCD22+ disease and one had emergence of MRD level disease, in all cases associated with loss of CAR T cells. One other patient had early loss of CAR T cells with B cell recovery but ongoing MRD negative CR at 3 months post-infusion and remains in MRD negative CR on maintenance chemotherapy. Overall, at a median follow-up of 8.7 months (range 1-15 months), 6/10 responding patients remain in MRD negative CR at last follow-up. Importantly, antigen-negative relapse has not been observed. Summary/Conclusion: Our data show that dual CD19/22 targeting CAR T cells generated by co-transduction show a favorable safety profile, with robust expansion/persistence and early efficacy in a heavily pre-treated cohort. To date with we have not observed antigen negative relapse. This contrasts to an incidence of 5/6 CD19 negative relapses in the 12 responders treated with single CD19 targeting CAR T cells (AUTO1) in an earlier cohort and suggests dual targeting may be effective in preventing antigen escape. However, longer follow up will be needed to confirm this. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A significant proportion of patients suffering from acute myeloid leukemia (AML) cannot be cured by conventional chemotherapy, relapsed disease being a common problem. Molecular targeting of essential oncogenic mediators is an attractive approach to improving outcomes for this disease. The hematopoietic transcription factor c-MYB has been revealed as a central component of complexes maintaining aberrant gene expression programs in AML. We have previously screened the Connectivity Map database to identify mebendazole as an anti-AML therapeutic targeting c-MYB. In the present study we demonstrate that another hit from this screen, the steroidal lactone withaferin A (WFA), induces rapid ablation of c-MYB protein and consequent inhibition of c-MYB target gene expression, loss of leukemia cell viability, reduced colony formation and impaired disease progression. Although WFA has been reported to have pleiotropic anti-cancer effects, we demonstrate that its anti-AML activity depends on c-MYB modulation and can be partially reversed by a stabilized c-MYB mutant. c-MYB ablation results from disrupted HSP/HSC70 chaperone protein homeostasis in leukemia cells following induction of proteotoxicity and the unfolded protein response by WFA. The widespread use of WFA in traditional medicines throughout the world indicates that it represents a promising candidate for repurposing into AML therapy.

Background: CD19 negative escape is a major cause of relapse after CD19 CAR T cell therapy for relapsed/refractory (r/r) paediatric ALL and dual targeting of CD19/CD22 may overcome this. We have previously shown that AUTO1, a fast off rate autologous CD19 CAR T cell therapy was highly active in ALL with a favorable safety profile and excellent persistence (Ghorashian et al Nat.Med. 2019). Building on these properties, we developed AUTO1/22 in which autologous T cells are co-transduced with 2 different lentiviral vectors encoding our existing CD19 CAR and a novel CD22CAR designed to recognise targets with low antigen density. AUTO1/22 was evaluated in a Phase I study in children/young adults with r/rALL (NCT02443831). Aims: To determine the safety/biological efficacy of AUTO1/22 Methods: Patients with r/r B-ALL age < 25 ywho were ineligible for/relapsed after Tisagenlecleucel were recruited. Following fludarabine/cyclophosphamide lymphodepletion, patients received 1x106 /kg CAR+ T cells. The presence of CAR T cells in the blood/bone marrow (BM) was assessed by flow cytometry + qPCR and BM MRD was assessed by IgH qPCR + flow cytometry. Primary end-points were incidence of grade 3-5 toxicity and the proportion of patients achieving MRD negative remission. Results: Ten patients have been treated and 8 are evaluable with >1 month follow-up. The median age was 12 years and patients had a median of 3.5 prior lines of therapy (range 2-6). Five of 8 patients had relapsed post allogeneic SCT, 4 had received prior Blinatumomab/Inotuzumab and 3 had relapsed after prior Tisagenlecleucel. Prior to lymphodepletion, 2 patients had >5% BM disease, 5 had MRD between 10-2 and 10-5 and 1 was BM MRD negative. CAR T cell products had a central memory phenotype with predominance of CD19/22 double positive cells (median 59.3%) and balanced populations of CD19 and CD22 single positive cells (16% and 10.9% respectively).Cytokine release syndrome (CRS) occurred in 7/8 patients (grade 1 n=2, grade 2 n=5) requiring Tocilizumab in 3 cases, but severe (≥ grade 3) CRS was not seen and no patients required ICU admission for CRS. Grade 1-2 ICANS was observed in 3 patients. One patient had delayed grade 4 leucoencephalopathy (MRI/brain biopsy were more indicative of fludarabine toxicity than CAR T related) and has ongoing neurological recovery. 7 patients had grade 3-4 cytopenia persisting beyond/recurring after day 28, requiring a CD34+ stem cell top up in 1 case. 5/8 patients had CD19CAR T cells and 3/8 patients had CD22CAR T cells detectable at last follow-up. 7 of 8 evaluable patients (88%) achieved MRD negative CR/CRi at 1 month post-infusion. One patient did not respond with CD19+ CNS relapse + MRD level BM disease at day 28. Of the 7 responding patients, 1 had frank CD19+CD22+ BM and extramedullary relapse at 3 months and 1 had emergence of MRD level disease at 10.5 months post infusion, in both cases associated with loss of CAR T cells. One other patient had early loss of CAR T cells with B cell recovery but ongoing MRD negative CR at 3 months post-infusion and remains in MRD negative CR on maintenance chemotherapy. Overall, at a median follow-up of 4.8 months, 5/8 patients remain in MRD negative CR at last follow-up. Summary/Conclusion: We demonstrate that dual CD19/22 targeting CAR T cells generated by co-transduction show an acceptable safety profile, with robust expansion/persistence and early efficacy in a heavily pre-treated cohort. To date with limited follow-up we have not observed antigen negative relapse but longer follow up is needed.

KMT2A- rearranged infant ALL is an aggressive childhood leukemia with poor prognosis. Here, we investigated the developmental state of KMT2A -rearranged infant B-cell acute lymphoblastic leukemia (B-ALL) using bulk messenger RNA (mRNA) meta-analysis and examination of single lymphoblast transcriptomes against a developing bone marrow reference. KMT2A -rearranged infant B-ALL was uniquely dominated by an early lymphocyte precursor (ELP) state, whereas less adverse NUTM1 -rearranged infant ALL demonstrated signals of later developing B cells, in line with most other childhood B-ALLs. We compared infant lymphoblasts with ELP cells and revealed that the cancer harbored hybrid myeloid–lymphoid features, including nonphysiological antigen combinations potentially targetable to achieve cancer specificity. We validated surface coexpression of exemplar combinations by flow cytometry. Through analysis of shared mutations in separate leukemias from a child with infant KMT2A -rearranged B-ALL relapsing as AML, we established that KMT2A rearrangement occurred in very early development, before hematopoietic specification, emphasizing that cell of origin cannot be inferred from the transcriptional state.

Transient abnormal myelopoiesis (TAM) is a pre-leukaemic syndrome associated with Trisomy 21 (Down syndrome). TAM typically regresses without treatment, but if needed TAM cells are exquisitely sensitive to a short course of low-dose cytarabine (LDAC). A significant minority of TAM patients progress to myeloid leukaemia of Down Syndrome (MLDS) requiring intensive chemotherapy. We describe a neonate with unusually recalcitrant TAM who required four 7-day courses of LDAC. Neonates with Down syndrome are at significant risk of severe toxicity from chemotherapy but this was mitigated by the use of repeated courses of LDAC, without the need for more intensive MLDS treatment.

Background Minimal residual disease (MRD) measured on end-of-induction bone marrow (BM) is the most important biomarker for guiding therapy in pediatric acute lymphoblastic leukemia (ALL). Due to limited sensitivity of current approaches, peripheral blood (PB) is not a reliable source for identifying patients needing treatment changes. We sought to determine if high-throughput sequencing (HTS) (next-generation sequencing) of rearranged immunoglobulin and T-cell receptor genes can overcome this and be used to measure MRD in PB. Procedure We employed a quantitative HTS approach to accurately measure MRD from one million cell equivalents of DNA from 17 PB samples collected at day 29 after induction therapy in patients with precursor B-cell ALL. We compared these results to the gold-standard real-time PCR result obtained from their paired BM samples, median follow-up 49 months. Results With the increased sensitivity, detecting up to one abnormal cell in a million normal cells, we were able to detect MRD in the PB by HTS in all those patients requiring treatment intensification (MRD >= 0.005% in BM). Conclusion This is proof of principle that using the increased sensitivity of HTS, PB can be used to measure MRD and stratify children with ALL. The method is cost effective, rapid, accurate, and reproducible, with inherent advantages in children. Importantly, increasing the frequency testing by PB as opposed to intermittent BM sampling may allow extension of the dynamic range of MRD, giving a more complete picture of the kinetics of disease remission while improving relapse prediction and speed of detection.

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.

Mixed phenotype acute leukaemia (MPAL) is rare, comprising <5% of paediatric acute leukaemia.1 Prospective clinical trials are lacking, therefore there is no consensus about optimal treatment, although recent surveys by the United States Childhood Oncology Group (COG) and iBFM AMBI2012 registry have established that lymphoid directed treatment is associated with superior outcomes.1, 2 This is especially the case for patients with CD19-positive B-myeloid leukaemia if they obtain a good flow or molecular minimal residual disease (MRD) response to standard acute lymphoblastic leukaemia (ALL) induction therapy. MRD monitoring in MPAL can be challenging due to the mixed phenotype (flow) and potential escape of myeloid sub-clones under selection pressure of lymphoid directed therapy, which may or may not carry clonal immunoglobulin/T-cell receptor (Ig/TCR) rearrangements (molecular).3-5 While nearly 70% of patients with MPAL achieve flow MRD-negative remission at the end of ALL induction, those who do not achieve this have a significantly worse event-free survival. Such patients are often given myeloid therapy as bridge to haemopoietic stem cell transplant (HSCT) but the success of such an approach relies on obtaining a good MRD response without excess toxicity, which is often difficult to achieve. Hence, a more effective and less toxic approach might be to target CD19 antigen expressed on the majority blast cell population in patients with B-myeloid MPAL using blinatumomab. Blinatumomab a CD3/CD19 bi-specific T-cell engager is effective not only in relapse/refractory precursor B-cell ALL6 but also histone-lysine N-methyltransferase 2A (KMT2A) rearranged infant ALL, where a significant proportion of patients have aberrant expression of myeloid antigens on blasts.7, 8 Furthermore, blinatumomab has been used to good effect in adults with MPAL and CD19-positive AML as a bridge to transplant or consolidation chemotherapy/donor lymphocyte infusion.9-11 Here, we report on blinatumomab as an effective bridge to transplant in three paediatric patients with MPAL, including one that arose as a consequence of ALL therapy. All three patients had separate populations of different lineages immunophenotypically as opposed to a single clone with co-expression of lymphoid and myeloid markers (shown along with other patient characteristics in Table I). MRD was assessed by flow cytometry and either Ig/TCR gene or KMT2A real-time polymerase chain reaction (PCR) to a sensitivity of 1E-5, shown in Table II. P1 Mono CD19+ 85% P2 ProB CD19+ 15% P1 Mono CD19+ 8·5% P2 ProB CD19+ 1·5% (Ig/TCR MRD 2%) P1 Mono CD19+ 0·22% P2 ProB CD19+ 0·06% (Ig/TCR MRD 0·06%) P1 Mono CD19+ 0% P2 ProB CD19+ 0% (Ig/TCR MRD undetectable) P1 B-ALL CD19i+ 37% P2 Biphen CD19+ 21% P3 Mono CD19− 42% P1 B-ALL CD19+ 2·3% P2 Biphen CD19+ 2·9% P3 Mono CD19− 6·1% (Ig/TCR MRD 1%) P1 B-ALL CD19+ 0·0024% P2 Biphen CD19+ 0% P3 Mono CD19− 0% (Ig/TCR MRD 0·5%) P1 B-ALL CD19+ 0% P2 Biphen CD19+ 0% P3 Mono CD19− 0% (Ig/TCR MRD undetectable) P1 ProB CD19+ 7% P2 Biphen CD19+ 64% P3 Mono CD19+ 29% P1 ProB CD19+ 0·16% P2 BiPhen CD19+ 9·8% P3 Mono CD19+ 9·3% (KMT2A MRD not done) P1 ProB CD19+ 0% P2 BiPhen CD19+ 0% P3 Mono CD19+ 0% (KMT2A MRD 4%) P1 ProB CD19+ 0% P2 BiPhen CD19+ 0% P3 Mono CD19+ 0% (KMT2A MRD 1·8%) Patient 1 had persistent disease with predominantly myeloid phenotype at the end of ALL induction, therefore was initially given myeloid directed therapy [fludarabine, high-dose cytarabine and idarubicin (FLA-IDA)] but remained MRD positive with CD19 expressing blasts. Hence, blinatumomab was used as consolidation pre-HSCT after which she was in a flow and molecular MRD-negative remission and remains so 2 years after a fludarabine, treosulfan, thiotepa (FTT) conditioned 9/10 1A mismatched sibling HSCT. Patient 2 had >10% residual disease after ALL induction representing all three phenotypic compartments present at diagnosis including both CD19-positive and -negative components. Blinatumomab salvage resulted, surprisingly, in complete eradication of the CD19-negative component with persistent very low level (MRD 0·002%) CD19-positive disease, which responded to FLA-Ida prior to HSCT. He remains in remission 8 months after a FTT conditioned 10/10 matched unrelated HSCT with 100% donor chimerism. Patient 3, who had treatment-related MPAL, also responded to blinatumomab after debulking ALL chemotherapy and achieved a flow negative remission but had persistent molecular KMT2A-rho guanine nucleotide exchange factor 12 (ARHGEF12) MRD. She received high-dose cytarabine (HiDAC) consolidation and has full donor engraftment with negative molecular and flow MRD 5 months after FTT conditioned 10/10 matched sibling HSCT. We report MRD responses to blinatumomab in three patients with a mixed phenotype that included a significant CD19-negative compartment. All three were able to proceed to HSCT and remain in remission 5 months to 2 years after the procedure. Blinatumomab was well tolerated with mild to moderate cytokine release syndrome, which required steroids and brief interruption of infusion in patient 1 only. All patients were able to be discharged from hospital after the first week of therapy and received their infusions as outpatient. Interestingly, blinatumomab completely eradicated the CD19-negative (myeloid) component of disease in patient 2. We can only speculate as to the mechanism. Did a cytokine storm triggered by blinatumomab engaging with CD19-positive disease lead to a 'bystander kill' of the CD19-negative clones? Mechanistic analysis in models of solid tumours and haematological malignancies suggests that cytokines released by blinatumomab-activated T cells induces upregulation of bystander cells, contributing to T-cell induced bystander cell lysis, in presence of the specific antigen targeted.12, 13 A more mundane alternative explanation is that the apparently CD19-negative component had antigen expression below the threshold of detection by flow cytometry but within blinatumomab's capacity to do so. This approach achieved deep molecular responses resulting in achievement of normal bone marrow function with less toxicity than using conventional chemotherapy. Although one of the three patients remained low-level PCR MRD-positive before HSCT, the negative flow MRD indicates that the PCR was detecting non-proliferating primitive leukaemic stem cells, which may not be a good target for blinatumomab. In conclusion, blinatumomab is safe and highly effective in children with resistant/refractory B-myeloid MPAL as a bridge to transplant. Due to novelty of this approach and potential concerns about lineage switch or myeloid escape due to selective pressure on the CD19-positive component, all the patients reported here were given myeloid directed consolidation prior to HSCT. Whether this is required will need to be tested in future clinical trials. Jack Bartram and Ajay Vora designed the research and wrote the paper. Milena Balasch-Carulla, Shashank Bhojaraja and Nimish Kulkarni collected data and wrote the paper. Stuart Adams and Sarah Inglott performed all diagnostic characterisation. Olya O'Connor and Arunthethy Mahendrayogam wrote the paper. Danny Cheng and Vesna Pavasovic contributed clinical information. The authors have no conflicts to disclose.

Comparison of intratumor genetic heterogeneity in cancer at diagnosis and relapse suggests that chemotherapy induces bottleneck selection of subclonal genotypes. However, evolutionary events subsequent to chemotherapy could also explain changes in clonal dominance seen at relapse. We therefore investigated the mechanisms of selection in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) during induction chemotherapy where maximal cytoreduction occurs. To distinguish stochastic versus deterministic events, individual leukemias were transplanted into multiple xenografts and chemotherapy administered. Analyses of the immediate post-treatment leukemic residuum at single-cell resolution revealed that chemotherapy has little impact on genetic heterogeneity. Rather, it acts on extensive, previously unappreciated, transcriptional and epigenetic heterogeneity in BCP-ALL, dramatically reducing the spectrum of cell states represented, leaving a genetically polyclonal but phenotypically uniform population, with hallmark signatures relating to developmental stage, cell cycle and metabolism. Hence, canalization of the cell state accounts for a significant component of bottleneck selection during induction chemotherapy. Enver and colleagues report that epigenetic cell state, rather than genetic diversity, drives bottleneck selection of subclonal genotypes during induction chemotherapy in childhood B-cell precursor acute lymphoblastic leukemia.

Abstract Background 'Off-the-shelf' CAR T cell therapies are being investigated as alternatives to autologous CAR therapy, and can be generated using genome editing from allogeneic donors. Strategies to address HLA barriers include disruption of T cell receptor expression to prevent GVHD and removal of cell surface HLA expression to obviate host mediated rejection, as well as removal of CD52 to create a survival advantage in the presence of Alemtuzumab. In this study, lentiviral mediated CAR19 expression is linked to CRISPR editing through the incorporation of CRISPR guide RNA sequences within the vector 3'Long terminal repeat (LTR). The trial is in progress using pre-manufactured batches of TT52CAR19 T cells. Investigational medicinal product (IMP) Three allogeneic non-HLA matched donor derived CAR19 T cell banks were manufactured from steady state apheresis harvests from registry volunteer donors, using a semi-automated process under compliant conditions. Cells were activated with anti-CD3/CD28 (Transact) reagent and transduced with a lentiviral vector, TT52CAR19, and dual guide sgRNA cassettes targeting the T cell receptor alpha chain (TRAC) and CD52 loci. Next electroporation of Cas9 mRNA elicited transient genome editing, and automated magnetic bead depletion removed remaining TCRαβ+ T cells (mean 0.7%) and enriched CAR19+ T cells (mean 92.8%). Cells were cryopreserved in aliquots suitable for dose-banding and subjected to release testing, including flow cytometry, quantification of copy number (mean 3.83) and exclusion of replication competent lentivirus. Potency of each bank was confirmed in human:murine chimera experiments. Trial sponsorship & approvals The study is open at a single site and is sponsored by Great Ormond Street Hospital NHS trust and is supported by the Medical Research Council and National Institute for Health Research. Clinical trial authorisation was awarded by the MHRA after expert review, and ethical approval including gene therapy advisory committee (GTAC) review, and health research authority (HRA) approval. The study is open label, single arm and non-randomised. Study aims and Objectives The study aims to establish the safety and feasibility of TT52CAR19 for the induction of molecular remission in children with relapsed /refractory CD19-positive B-cell acute lymphoblastic leukaemia (B-ALL) within 28 days, ahead of planned allogeneic haematopoietic stem cell transplantation (allo-SCT). Assessments include time to remission, duration of remission, disease-free survival and overall survival. Expansion, persistence and elimination of TT52CAR19 cells and tracking of immune recovery after allo-SCT is monitored. Recording of complications and toxicities, including possible genotoxic side effects from CRISPR/Cas9 modification provides safety profile information. Eligibility, infusion and outcomes The study plans to treat 10 children aged between 6 months and < 18 years with CD19+ B-ALL quantified at >10 -4 in marrow (by flow or PCR) who are ineligible for autologous therapy. To date 2/4 children screened were found eligible and proceeded to lymphodepletion comprising Fludarabine, Cyclophosphamide and Alemtuzumab followed by a single infusion of 0.8-2.0x10 6 CAR19 T cells and a maximum of 5x10 4/kg TCRαβ T cells. In both cases, there was no GVHD or CRS >grade 1 and recovery of neutrophil counts by d28. Molecular remission was achieved in one child, where TT52CAR19 cells persistence was tracked by chimerism and copy number until further conditioning and allo-SCT. This child remains in remission >6 months later. Conclusions Feasibility of pre-manufacturing off-the-shelf CRISPR/Cas9 edited CAR19 T cells is demonstrated and the trial has provided first in human safety data and preliminary indications of potent anti-leukaemic activity in one of two subjects dosed. Disclosures Qasim: Autolus: Current equity holder in publicly-traded company; Novartis: Honoraria; Servier: Research Funding; Tessa: Membership on an entity's Board of Directors or advisory committees.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive bone marrow malignancy that accounts for ; 10% to 15% of pediatric acute lymphoblastic leukemia (ALL) and 20% of adult ALL. 1,2 Despite improvements in frontline treatments, outcomes in primary refractory (PR) or relapsed T-ALL remain dismal, and novel therapies are urgently needed. 3-5 In relapsed B-cell ALL, chimeric antigen receptor (CAR) T cells targeting CD19 have shown promising results and have recently been approved by the US Food and Drug Administration. In contrast, identifying suitable CAR T-cell targets against T-ALL has been far more challenging. Because CAR T cells share antigens with malignant T cells, mutual killing of CAR T cells, termed “ fratricide, ” can occur, preventing the generation, expansion, and persistence of CAR T cells. 6 Furthermore, unlike B-cell aplasia, which is largely manageable, prolonged and profound T-cell aplasia is intolerable and can result in life-threatening opportunistic infections. 7 Therefore, the ideal target would be one that is highly expressed on the

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Although there have been tremendous advances in the successful treatment of paediatric ALL, this disease contributes to half of all leukaemia deaths in children. Development of new, broad range therapies is urgently needed. In this project we focused our attention on STAT3 in B-ALL, investigating the STAT3 regulated pathways. Global gene expression changes in B-ALL REH cells following pharmacological inhibition and shRNA-mediated silencing of STAT3 highlighted induction of TP53 target genes, without significant changes in TP53 mRNA. Indeed, STAT3 inhibition led to increased TP53 protein levels and to increased expression of TP53-target genes. Loss of TP53 expression in CRISPR/Cas9-generated TP53-/- REH cells resulted significant attenuation of REH sensitivity to STAT3 inhibition. We have also discovered that susceptibility to STAT3 inhibition is much broader in patient-derived xenograft (PDX) B-ALL subtypes than previously noted in cell lines, and that it correlates with TP53 status. Our results indicate a functional link between STAT3 and TP53 in B-ALL. STAT3 inhibition coupled with TP53 induction could represent a novel therapeutic strategy in B-ALL.

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.