MLL-rearranged Acute Lymphoblastic Leukemia Research Articles

Bispecific CAR-iNKT Immunotherapy for High Risk MLL-Rearranged Acute Lymphoblastic Leukemia

MLL-rearranged CD19+ acute lymphoblastic leukemia ( MLLr-ALL), the commonest infant leukemia, has a high mortality rate (<40% leukemia-free survival). While stem cell transplantation has not improved outcomes, CD19-directed immunotherapeutic approaches, including CAR-T cells, have shown promise. However, as patients can still relapse with CD19-, or lineage-switched disease, alternative strategies are needed. To enhance efficacy and mitigate against immune escape, we aimed to develop iNKT cell-based bispecific CAR immunotherapy. iNKT are rare, CD1d-restricted, glycolipid-reactive T cells that bridge adaptive and innate immunity. Furthermore, allogeneic iNKT protect from aGVHD making them ideal candidates for ‘off-the-shelf’ immunotherapy without need for TCR deletion. To target both CD19 and CD133, which is often co-expressed with CD19 on MLLr blasts and possibly also on CD19-CD133+ leukemia-initiating cells, we developed a CD19-28z/CD133-41BBz bispecific as well as CD19-28z and CD133-4-1BBz monospecific CARs and compared their anti-leukemic activity. In short- and long-term cytotoxicity assays against MLLr leukemic cell lines (SEM, RS4;11, KOPN8) we show that bispecific CAR-iNKT are more potent than their monospecific counterparts. In assays involving single or dual knock-out of CD19 and CD133 in SEM cells, both the CD19 and CD133 moieties of the bispecific CAR were reactive and specific against their respective targets. While bispecific CAR-iNKT retained killing activity against either single target-expressing leukemia cells, monospecific CAR-iNKT only killed leukemia cells expressing their cognate target. In line with the in vitro findings, in xenograft assays, a low dose of 10 6 bispecific CAR-iNKT cells significantly prolonged survival of luc-SEM leukemia-bearing mice as compared to monospecific CAR-iNKT-treated and untreated control animals (Fig 1A). Of note, in vivo as well as in vitro, bispecific CAR-iNKT very effectively eliminated high and low CD19/CD133 co-expressing, but not, as expected, CD19-CD133- disease. Next, we compared bispecific CAR-iNKT vs their CAR-T counterparts. In vitro, CAR-iNKT from three donors demonstrated higher killing activity than CAR-T, while in vivo, compared to 10 6 CAR-T, CAR-iNKT cells induced significantly improved survival of leukemia-bearing mice (Fig 1B). Of note, while CAR-T cells were not detectable in the BM and spleen at sacrifice, CAR-iNKT were readily detectable in the organs of CAR-iNKT cell-treated animals, suggesting they persist longer. Remarkably, mice treated with 5x10 6 bispecific CAR-iNKT cells survived >60 days without any evidence of leukemia by serial bioluminescence assay, or in the bone marrow, spleen (flow-cytometry) and leptomeninges (immunohistochemistry) upon culling. Since SEM cells do not express CD1d, we explored alternative mechanism(s) that would account for the more potent anti-leukemic activity of CAR-iNKT cells. We found that CAR-iNKT express significantly higher baseline levels of the activating receptor NKG2D than CAR-T cells. Upon co-culture with SEM cells, nearly all CAR-iNKT but only a fraction of CAR-T express NKG2D, and a blocking NKG2D Ab significantly abrogated the superior cytotoxic effect of CAR-iNKT cells. Consistent with an NKG2D-dependent mechanism, mRNA of one or more NKG2D ligands is expressed by primary MLLr blasts as well as leukemia cell lines. Since CD133 is expressed on hematopoietic stem and progenitor cells (HSPC), the potential hematopoietic toxicity of bispecific CAR-iNKT cells was tested in a series of experiments involving cord blood humanisation of NSGS mouse hematopoiesis. We found that transfer of 10 7 bispecific CAR-iNKT cells significantly but transiently depleted engrafted CB CD19+ B cells. However, phenotypic analysis showed that long term engraftment levels were similar for all mature cell lineages, CD34+ HSPC and specifically for Lin-CD34+CD38- HSC. We conclude that bispecific CAR-iNKT cell immunotherapy is a very effective treatment for pre-clinical aggressive MLLr-ALL, it outperforms CAR-T cell immunotherapy in an NKG2D-dependent manner and has the potential to protect from immune escape, leptomeningeal disease and lineage switch without discernible hematological toxicity. These findings provide the basis for clinical development of bispecific CD19/CD133 CAR-iNKT cells as an ‘off-the-shelf’ treatment for MLLr-ALL.

Targeting HDAC9 Contributes to Degradation of MLL Fusion Oncoproteins in KMT2A-Rearranged Acute Myeloid Leukemia

Acute myeloid leukemia with MLL rearrangement (MLLr-AML) is a highly aggressive subtype of AML. Existing treatment strategies are nonspecific and ineffective. The MLL fusion gene is one of the driver oncogenes in leukemia and is central to the pathogenesis of MLLr-AML, and more research has recently begun to focus on targeting MLL fusion proteins. Targeting MLL fusion proteins has been shown to have a tumor suppressor effect, demonstrating efficacy in preclinical models of MLLr-AML, and in 2022 Scott A Armstrong ‘s team suggested that degradation of MLL-AF9 can cause an increase in cell differentiation and apoptosis, confirming the efficacy of targeting MLL fusions. However, specific targets that degrade MLL fusion proteins as well as clinically translatable targeted therapies are still lacking and need to be further explored. Study confirmed that MLL-rearranged ALL cells are highly sensitive to the broad-spectrum HDAC inhibitor panobinostat. A recent clinical study by Mireia Camós’ team proposed that HDAC9 has a significantly high expression in childhood MLL-rearranged B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and AML, but that article did not address any mechanistic studies. The potential value of HDAC9 in adult lymphoid hematologic diseases has been gradually revealed, however, the expression profile and precise molecular mechanisms of HDAC9 in adult myeloid hematologic tumors, have not been reported, which deserves to be further explored. In order to investigate the expression and role of HDAC9 in MLLr-AML, we analyzed the second-generation sequencing data from the Beat AML dataset and found that HDAC9 has a significantly high expression in MLLr-AML, while only HDAC9 is characterized by this feature in HDAC family molecules. Further we analyzed the data of 300 adult AML specimens in our hospital (including 20 MLLr-AML patients), and the results further confirmed that only HDAC9 among the HDAC family had a significantly higher expression level in the MLLr-AML patient group than that in the Non-MLLr-AML patient group (P<0.001), and the high expression of HDAC9 in the MLLr-AML patient group was a common phenomenon and not limited to a particular class of MLL chaperone molecules. Further studies revealed that overexpression of MLL fusion proteins in HEK293T could cause up-regulation of HDAC9 protein levels, and this experiment clarified the direct correlation between the MLL fusion genes and the high expression of HDAC9. The HDAC IIa inhibitor (TMP-269), which can target HDAC9, was significantly more sensitive in MLLr-AML cell lines than in Non-MLLr-AML cell lines, and also that MLLr-AML cell survival was dependent on HDAC9, and that knockdown of HDAC9 induced an increase in apoptosis and a slowdown in proliferation in MLLr-AML cells. We subsequently demonstrated that knockdown of HDAC9 downregulates the expression of MLL fusion proteins through degradation of MLL proteins. Furthermore, when exploring therapeutic options for targeting MLLr-AML, we found that combining the BCL-2 inhibitor Venetoclax (VEN) with an MLL-Menin specific inhibitor (MEN1i) specifically downregulated the expression of HDAC9 and had a favorable synergistic killing effect on MLLr-AML in both in vitro and in vivo models, further suggesting an important role of HDAC9 in the treatment of MLLr-AML. To sum up, through this study, we can shed light on the role of specific HDAC molecules in MLLr-AML and provide a new potential target for the degradation of MLL fusion protein to eradicate MLLr-AML.

Constitutively Synergistic Multiagent Drug Formulations Targeting MERTK, FLT3, and BCL-2 for Treatment of AML.

Although high-dose, multiagent chemotherapy has improved leukemia survival rates, treatment outcomes remain poor in high-risk subsets, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) in infants. The development of new, more effective therapies for these patients is therefore an urgent, unmet clinical need. The dual MERTK/FLT3 inhibitor MRX-2843 and BCL-2 family protein inhibitors were screened in high-throughput against a panel of AML and MLL-rearranged precursor B-cell ALL (infant ALL) cell lines. A neural network model was built to correlate ratiometric drug synergy and target gene expression. Drugs were loaded into liposomal nanocarriers to assess primary AML cell responses. MRX-2843 synergized with venetoclax to reduce AML cell density in vitro. A neural network classifier based on drug exposure and target gene expression predicted drug synergy and growth inhibition in AML with high accuracy. Combination monovalent liposomal drug formulations delivered defined drug ratios intracellularly and recapitulated synergistic drug activity. The magnitude and frequency of synergistic responses were both maintained and improved following drug formulation in a genotypically diverse set of primary AML bone marrow specimens. We developed a nanoscale combination drug formulation that exploits ectopic expression of MERTK tyrosine kinase and dependency on BCL-2 family proteins for leukemia cell survival in pediatric AML and infant ALL cells. We demonstrate ratiometric drug delivery and synergistic cell killing in AML, a result achieved by a systematic, generalizable approach of combination drug screening and nanoscale formulation that may be extended to other drug pairs or diseases in the future.

Telomere Transcription in MLL-Rearranged Leukemia Cell Lines: Increased Levels of TERRA Associate with Lymphoid Lineage and Are Independent of Telomere Length and Ploidy

Telomere transcription into telomeric repeat-containing RNA (TERRA) is an integral component of all aspects of chromosome end protection consisting of telomerase- or recombination-dependent telomere elongation, telomere capping, and the preservation of the (sub)telomeric heterochromatin structure. The chromatin modifier and transcriptional regulator MLL binds to telomeres and regulates TERRA transcription in telomere length homeostasis and response to telomere dysfunction. MLL fusion proteins (MLL-FPs), the product of MLL rearrangements in leukemia, also bind to telomeric chromatin. However, an effect on telomere transcription in MLL-rearranged (MLL-r) leukemia has not yet been evaluated. Here, we show increased UUAGGG repeat-containing RNA levels in MLL-r acute lymphoblastic leukemia (ALL) when compared to non-MLL-r ALL and myeloid leukemia. MLL rearrangements do not affect telomere length and UUAGGG repeat-containing RNA levels correlate with mean telomere length and reflect increased levels of TERRA. Furthermore, high levels of TERRA in MLL-r ALL occur in the presence of telomerase activity and are independent of ploidy, an underestimated source of variation on the overall transcriptome size in a cell. This MLL rearrangement-dependent and lymphoid lineage-associated increase in levels of TERRA supports a sustained telomere transcription by MLL-FPs that correlates with marked genomic stability previously reported in pediatric MLL-r ALL.

The RNA Binding Protein Musashi-2 (MSI2) Sustains the Growth and the Leukemogenic Potential of MLL-Rearranged Acute Lymphoblastic Leukemia, and It Is Involved in Glucocorticoid Resistance

Acute Lymphoblastic Leukemia occurring in infants and carrying the rearrangement of KMT2A, aka MLL (MLLr infant ALL) is a rare but very aggressive leukemia typically associated with poor outcome. Patients suffer from a dismal prognosis (overall survival: 20-40%) mainly due to the resistance to conventional therapy and a high incidence of relapse. The biological mechanisms involved in the pathogenesis and disease progression of infant MLLr leukemia are not completely understood and the identification of druggable genes is urgently needed for the development of novel therapeutic strategies. The RNA-binding protein Musashi-2 (MSI2) is a post-transcriptional regulator of mRNA translation to proteins. MSI2 has a pivotal role in normal hematopoiesis and in a variety of tumors, including leukemia, where it is involved in cell proliferation, differentiation and maintenance of the (cancer) stem cell pool. Although several studies strikingly demonstrated the involvement of MSI2 in chronic and acute myeloid leukemia, with regards to B-ALL, few papers reported over-expression of MSI2 associated with a poor outcome in both pediatric and adult B-ALL patients; however functional data are still missing. By retrospectively analyzing the gene expression profile of a large cohort of Italian patients with pediatric leukemia (n=314) we observed that MSI2 is highly expressed across the different cytogenetic subgroups, and the level of expression is even higher in ALL compared to AML cases; thus reinforcing the hypothesis that MSI2 might have a crucial role also in ALL. To unravel the functional role of MSI2 in MLL-r ALL, we successfully generated a model of MSI2 knock-out (MSI2 KO) using CRISPR/CAS9 genome editing in the MLL::AF4+ ALL cell line SEM. In a long-term competitive assay in vitro, in which the MSI2 KO and CNTRL cells were co-cultured at the initial 90:10 ratio (KO:CNTRL), we observed that after 20 passages in culture the CNTRL counterpart overgrew the MSI2 KO cells (which indeed extinguished); thus demonstrating that the lack of MSI2 leads to a proliferation disadvantage. In agreement with this, MSI2 KO cells show an impaired leukemia-initiating capacity and a lower disease burden in vivo, and mice transplanted with MSI2 KO cells display a prolonged survival compared to controls. Also, we performed a high-throughput drug screening using a manually curated library of 174 compounds (FDA-approved, currently used in the clinic) and we observed that MSI2 KO cells show a marked sensitivity to Glucocorticoids (GCs). Further validation studies in vitro demonstrated that MSI2 KO cells were significantly (100-fold) more sensitive to Prednisolone or Dexamethasone than their MSI2-expressing counterpart. In addition, the treatment of SEM cells (typically resistant to GCs) with the MSI2 inhibitor Ro 08-2750 phenocopied the sensitization to GCs observed in CRISPR-edited MSI2 KO cells, showing synergistic effects in combination with Prednisolone or Dexamethasone. As infant MLLr ALL patients are typically resistant to GCs, our findings suggest that targeting MSI2 might represent a potential novel approach to circumvent the therapy resistance. Finally, by further investigating the biological mechanisms of GC-sensitization, we unraveled a potential role for MSI2 in the regulation of the energetic metabolism of leukemic cells. Indeed, the confocal analysis of mitochondrial membrane potential and the profiling of cellular bioenergetics by Seahorse technology revealed that MSI2 KO cells display mitochondrial hyperactivity at steady-state compared to controls. Upon GCs exposure (which inhibits glycolysis in both MSI2 KO and CNTRL cells) mitochondrial respiration is increased in CNTRL cells to compensate ATP production, while it is severely impaired in MSI2 KO, leading to a mitochondrial crisis and energy failure. The importance of metabolic plasticity of tumor cells in adaptation to stress conditions is emerging as a crucial aspect of cancer biology. The putative role of MSI2 in regulating the energy metabolism of leukemia is a novel finding not previously investigated, which may pave the way for targeting the metabolic vulnerabilities of ALL as a novel therapeutic approach. In conclusion, our study demonstrates that MSI2 sustains the growth in vitro and the leukemogenic potential in vivo of MLLr ALL cells, and it is involved in GC-resistance by regulating the energy metabolism of leukemia.

Abstract 681: Targeted MLL-AF9 degradation is phenocopied by combined DOT1L and Menin inhibition

Abstract MLL1 (KMT2A) translocations lead to aberrant expression of stem cell associated gene programs in hematopoietic cells producing a particularly aggressive subtype of leukemia, namely MLL-rearranged acute myeloid and lymphoblastic leukemia (AML and ALL, respectively). In pediatric leukemia, the most common rearrangement is the t(9; 11) (p22; q23) reciprocal translocation, which results in the expression of the MLL-AF9 fusion gene. Since the MLL-AF9 rearrangement does not lead to direct activation of an enzyme, small molecule drug discovery remains challenging and despite decades of research on MLL-rearranged leukemia, we have been limited by the inability of acutely assessing the consequences of direct MLL-AF9 inactivation in relevant model systems. By coupling rapid PROTAC-mediated degradation of the oncogenic MLL-AF9 fusion protein with genome-wide gene expression and chromatin analyses we have now been able to identify the immediate transcriptional and chromatin state consequences of MLL-AF9 degradation. Specifically, we have established a core set of MLL-AF9 target genes whose expression changes within minutes of MLL-AF9 degradation. These rapid expression changes are mediated by changes in productive RNA Polymerase II elongation and increased RNA Polymerase II pausing. At later timepoints, degradation of the fusion protein also induces loss of an active chromatin landscape at MLL-AF9 target genes, characterized by loss of MLL-AF9 associated proteins and activating histone modifications. These insights improve our mechanistic understanding of how MLL-AF9 mediates chromatin remodeling and leukemogenesis, while also helping us evaluate small molecule inhibitors of epigenetic mechanisms that target the MLL-AF9 protein complex. Specifically, we investigated how degradation of MLL-AF9 compares to enzymatic inhibition of DOT1L or inhibition of the MENIN-MLL interaction. Interestingly, both DOT1L and MENIN inhibitors fell short when comparing their effects to MLL-AF9 degradation and only combined DOT1L/MENIN inhibition induced global MLL-AF9 destabilization on chromatin, thus disrupting the full MLL-AF9 directed epigenetic and transcriptional program. Ultimately, we were able to confirm these findings in a patient-derived xenograft model in vivo, where only the combination was able to significantly prolong leukemia free survival. In summary, our studies establish the cellular and molecular consequences of targeted degradation of the MLL-AF9 fusion oncoprotein and help improve our understanding of small molecule inhibitors of epigenetic mechanisms as cancer therapies. Citation Format: Sarah Naomi Olsen, Laura Godfrey, James P. Healy, Charles Hatton, Scott A. Armstrong. Targeted MLL-AF9 degradation is phenocopied by combined DOT1L and Menin inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 681.

CN470 is a BET/CBP/p300 multi-bromodomain inhibitor and has an anti-tumor activity against MLL-rearranged acute lymphoblastic leukemia

Acute lymphoblastic leukemia with chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene (MLL-r ALL) remains an incurable disease. Thus, development of a safe and effective therapeutic agent to treat this disease is crucial to address this unmet medical need. BRD4, a member of the bromodomain and extra-terminal domain (BET) protein family, and cyclic AMP response element binding protein binding protein (CBP) and p300, two paralogous histone acetyltransferases, are all considered cancer drug targets and simultaneous targeting of these proteins may have therapeutic advantages. Here, we demonstrate that a BET/CBP/p300 multi-bromodomain inhibitor, CN470, has anti-tumor activity against MLL-r ALL in vitro and in vivo. CN470, potently inhibited ligand binding to the bromodomains of BRD4, CBP, and p300 and suppressed the growth of MLL-r ALL cell lines and patient-derived cells with MLL rearrangements. CN470 suppressed mRNA and protein expression of MYC and induced apoptosis in MLL-r ALL cells, following a cell cycle arrest in the G1 phase. Moreover, CN470 reduced BRD4 binding to acetylated histone H3. The in vivo effects of CN470 were investigated using SEMLuc/GFP cells expressing luminescent markers in an orthotopic mouse model. Mice administered CN470 daily had prolonged survival compared to the vehicle group. Further, CN470 also showed anti-tumor effects against an MLL-r ALL patient-derived xenograft model. These findings suggest that inhibition of BET/CBP/p300 by the multi-bromodomain inhibitor, CN470, represents a promising therapeutic approach against MLL-r ALL.

Selective Inhibition of Class II HDAC Isoforms 4 and 5 Provides a Promising Therapeutic Intervention for MLL-Rearranged Acute Lymphoblastic Leukemia in Infants

▪MLL-rearranged acute lymphoblastic leukemia (ALL) is characterized by deregulation of the epigenome and shows susceptibility towards epigenetic perturbators such as histone deacetylase (HDAC) inhibitors. Hence, HDACs represent attractive drug targets and a variety of small molecule HDAC inhibitors have been developed and evaluated for the treatment of hematological malignancies. However, most broad-spectrum inhibitors, which simultaneously target the majority of human HDAC isoforms, often induce toxicity, especially in combination with other therapeutic agents. Therefore, selective inhibition of only one or two HDAC isoforms may represent a better alternative, provided that disease-specific dependency on specific HDACs has been identified.We examined the effects of shRNA-mediated knock-down of the class II HDACs (i.e. HDAC4, HDAC5, HDAC6, HDAC7 and HDAC9) in the MLL-rearranged ALL cell lines SEM and ALL-PO. Except for HDAC9, loss of expression (both on the mRNA and protein level) of all HDACs led to strong reductions in viable cells (0.70 to 0.19-fold; p=0.02-0.0016) in both models due to apoptosis, cell cycle arrest, or a combination thereof.Next, we evaluated the in vitro efficacy of a variety of class II HDAC-specific inhibitors on a panel of MLL-rearranged ALL (n=5) using 4-day viability MTT assays. This revealed that the selective HDAC4/5 inhibitor LMK-235 was able to recapitulate the loss-of-function phenotype of HDAC4 and HDAC5. Dose response curves showed complete growth inhibition in MLL-rearranged ALL cell lines (n=5), as well as in primary MLL-rearranged infant ALL patient samples (n=4), with IC 50 values of ~100 nM and 40-100 nM, respectively. Importantly, at these concentrations, LMK-235 hardly affected whole bone marrow samples derived from healthy individuals (n=2), for which IC 50 values were ~1 µM.To further explore the potential of class II HDAC inhibitor-based therapeutic strategies, we performed a combinatorial drug screen to identify compounds that synergize with LMK-235. For this, a compound library (comprising >200 unique agents) was screened in the absence and presence of varying concentrations of LMK235 in the MLL-rearranged cell line models SEM and ALL-PO. This, and subsequent validation experiments in additional cell line models, revealed that Venetoclax (BCL2 inhibitor), Trametinib (MEK/ERK inhibitor), Ponatinib (multi-tyrosine kinase inhibitor) and Omipalisib (a PI3K/mTOR inhibitor) strongly synergized with LMK-235. Average ZIP synergy scores ranged from 10-30, with peak ZIP scores up to 40. Importantly, synergistic effects were consistent over all concentration combinations tested. The addition of 50-100 nM LMK-235 strongly reduced IC 50 values for Omipalisib, Ponatinib and Venetoclax (0.27-fold p=0.003, 0.11-fold p=0.0005, 0.75-fold p=0.0004, respectively) in both models.In preparation to assess the in vivo efficacy of LMK-235 in patient-derived xenograft (PDX) mouse models of MLL-rearranged infant ALL, pharmacokinetic/pharmacodynamic (PK/PD) analysis was performed in immunodeficient NSG mice (n=5). For this, mice were treated with 20 mg/kg of LMK-235, daily administered via intraperitoneal injections for a total of 29 days. While none of the mice showed signs of toxicity or weight loss, LMK-235 plasma levels were stably maintained at concentrations that are highly effective against MLL-rearranged ALL cells in vitro.Taken together, these data demonstrate that various class II HDAC isoforms are targetable vulnerabilities in MLL-rearranged ALL and that pharmaceutical inhibition of HDAC4/5 by LMK-235 represents an attractive therapeutic option. Moreover, high levels of synergy observed between this HDAC inhibitor and various agents belonging to drug classes already reported to be effective against MLL-rearranged ALL, warrants pre-clinical evaluation in vivo. Currently, the assessment of the in vivo efficacy of LMK-235 monotherapy in MLL-rearranged infant ALL PDX models is in progress, after which promising synergistic HDAC inhibitor-based drug combinations will be evaluated. To determine the additional therapeutic value, the efficacy of LMK-235 and promising synergistic combinations will be evaluated in the background of conventional combination chemotherapy, where PDX models will receive a mouse-adapted version of induction therapy currently applied for treatment of MLL-rearranged infant ALL patients. DisclosuresNo relevant conflicts of interest to declare.

Safety and Efficacy of Menin Inhibition in Patients (Pts) with MLL-Rearranged and NPM1 Mutant Acute Leukemia: A Phase (Ph) 1, First-in-Human Study of SNDX-5613 (AUGMENT 101)

Safety and Efficacy of Menin Inhibition in Patients (Pts) with MLL-Rearranged and NPM1 Mutant Acute Leukemia: A Phase (Ph) 1, First-in-Human Study of SNDX-5613 (AUGMENT 101)

Identification and characterization of relapse-initiating cells in MLL-rearranged infant ALL by single-cell transcriptomics

Infants with MLL-rearranged infant acute lymphoblastic leukemia (MLL-r iALL) undergo intense therapy to counter a highly aggressive malignancy with survival rates of only 30–40%. The majority of patients initially show therapy response, but in two-thirds of cases the leukemia returns, typically during treatment. The glucocorticoid drug prednisone is established as a major player in the treatment of leukemia and the in vivo response to prednisone monotreatment is currently the best indicator of risk for MLL-r iALL. We used two different single-cell RNA sequencing technologies to analyze the expression of a prednisone-dependent signature, derived from an independent study, in diagnostic bone marrow and peripheral blood biopsies. This allowed us to classify individual leukemic cells as either resistant or sensitive to treatment and show that quantification of these two groups can be used to better predict the occurrence of future relapse in individual patients. This work also sheds light on the nature of the therapy-resistant subpopulation of relapse-initiating cells. Leukemic cells associated with high relapse risk are characterized by basal activation of glucocorticoid response, smaller size, and a quiescent gene expression program with cell stemness properties. These results improve current risk stratification and elucidate leukemic therapy-resistant subpopulations at diagnosis.

Reciprocal Regulation of BCL6 and MLL Expression As a Vulnerability in MLL -Rearranged Acute Lymphoblastic Leukemia

Background: Chromosomal translocations involving the Mixed Lineage Leukemia gene MLL account for ~70% of infant acute lymphoblastic leukemias (ALLs; Ayton and Cleary, 2001) and are associated with dismal prognosis. These findings underscore the importance of understanding the molecular mechanisms underlying disease progression of MLL-rearranged (MLLr) leukemia for the development of novel therapeutics. Recently, an integrative epigenomic analysis has identified the transcriptional repressor and proto-oncogene BCL6 as a transcriptional target in MLL-AF4 -driven pre-B ALL (Geng et al., 2012). BCL6 functions as a mediator of drug resistance to tyrosine kinase inhibitors in Ph+ ALL (Duy et al., 2011). Here, we tested the hypothesis that BCL6 is required for MLLr leukemia and represents a therapeutic target in MLLr pre-B ALL.Results: Western blot analysis of a panel of patient-derived pre-B ALL cells (MLLr : n= 7; ETV6-RUNX1 : n= 2; BCR-ABL1 : n= 1; not otherwise specified: n= 4) and normal CD19+ B cells (n= 3) revealed that BCL6 levels were elevated in MLL r ALL samples. Furthermore, immunohistochemistry staining in bone marrow biopsies from ALL patients (n= 72) showed that BCL6 levels were upregulated in MLLr cases. Expression of MLL-ENL or MLL-AF4 fusion protein increased Bcl6 protein levels (~10-fold) in murine pre-B cells. Moreover, ChIP-seq analysis of human MLLr pre-B ALL cell lines using antibodies against MLL, AF4 and ENL demonstrated direct binding to the BCL6 promoter. Studying methylation status revealed that the BCL6 promoter was hypomethylated in patients with MLLr pre-B ALL compared to patients with other subtypes of pre-B ALL (including BCR-ABL1, ETV6-RUNX 1 and hyperdiploid) as well as normal pre-B cells from healthy donors (St. Jude, n= 132, Figueroa, 2014). Consistent with these observations, multivariate analysis of high risk pre-B ALL patients showed that high BCL6 levels in combination with MLL rearrangements were associated with the worst clinical outcome (COG P9906). Collectively, our findings demonstrated that BCL6 levels are elevated in MLLr ALL, and that expression levels of MLL and BCL6 can serve as predictors for risk stratification in ALL.Interestingly, expression of wild-type MLL was positively correlated with that of BCL6 in both pediatric (COG P9906) and adult (ECOG E2993) pre-B ALL. Furthermore, multivariate analysis comparing MLLHighBCL6High patients with MLLLowBCL6Low patients showed that high expression levels of both genes correlated with poor clinical outcome (COG P9906). These findings prompted us to study whether MLL regulates BCL6 expression, and vice versa . Cre-mediated deletion of Mll abrogated imatinib-mediated induction of Bcl6 in BCR-ABL1 -driven pre-B ALL, indicating that Mll is required for upregulation of Bcl6 expression. While genetic inactivation of Bcl6 as well as treatment with the BCL6-specific retro-inverso peptide-inhibitor (RI-BPI) decreased Mll expression, doxycycline-induced expression of Bcl6 upregulated Mll expression. Combining chromatin immunoprecipitation and DNA microarray (ChIP-on-chip), we found that BCL6 was recruited to the MLL promoter in pre-B ALL. Taken together, these findings support our proposed scenario of reciprocal regulation of MLL and BCL6 expression.Notably, ChIP-seq analyses of MLLr pre-B ALL cells showed that there are 268 common gene targets of both MLL and BCL6. Among the common targets include cell cycle checkpoint regulators (CDKN1B, ETS2, BMI1, RB1), apoptosis facilitator BIM, and tumor suppressor TET2, indicating that BCL6 is a key regulator of the transcriptional program in MLLr pre-B ALL. In support of an essential role of BCL6 in MLLr ALL, expression of a dominant negative BCL6 mutant in patient-derived MLLr pre-B ALL cells induced rapid depletion from cell culture in competitive-growth assays. Furthermore, RI-BPI treatment resulted in cell cycle arrest, impaired colony forming ability and inhibited leukemia initiation in transplant recipient mice. Importantly, RI-BPI in combination with standard chemotherapy Vincristine exerted more potent effects on killing MLLr pre-B ALL cells than either agent alone.Conclusions: Our findings suggest that aberrant BCL6 expression levels are important for disease progression, and that pharmacological inhibition of BCL6 augments responsiveness to standard chemotherapy in the treatment of MLLr pre-B ALL. DisclosuresHurtz:Incyte: Research Funding. Armstrong:Cyteir: Consultancy; Imago: Consultancy; C4 Therapeutics: Consultancy; Epizyme: Consultancy.

Durable Remission after Salvage Chemotherapy with Combining Bortezomib and Vorinostat Followed By CD19 CAR-T Therapy in a Patient with MLL-Rearranged ALL after Early Relapse Post Allogeneic Hematopoietic Stem Cell Transplantation

Durable Remission after Salvage Chemotherapy with Combining Bortezomib and Vorinostat Followed By CD19 CAR-T Therapy in a Patient with MLL-Rearranged ALL after Early Relapse Post Allogeneic Hematopoietic Stem Cell Transplantation

Irinotecan Induces Disease Remission in Xenograft Mouse Models of Pediatric MLL-Rearranged Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) in infants (<1 year of age) remains one of the most aggressive types of childhood hematologic malignancy. The majority (~80%) of infant ALL cases are characterized by chromosomal translocations involving the MLL (or KMT2A) gene, which confer highly dismal prognoses on current combination chemotherapeutic regimens. Hence, more adequate therapeutic strategies are urgently needed. To expedite clinical transition of potentially effective therapeutics, we here applied a drug repurposing approach by performing in vitro drug screens of (mostly) clinically approved drugs on a variety of human ALL cell line models. Out of 3685 compounds tested, the alkaloid drug Camptothecin (CPT) and its derivatives 10-Hydroxycamtothecin (10-HCPT) and 7-Ethyl-10-hydroxycamtothecin (SN-38: the active metabolite of the drug Irinotecan) appeared most effective at very low nanomolar concentrations in all ALL cell lines, including models of MLL-rearranged ALL (n = 3). Although the observed in vitro anti-leukemic effects of Camptothecin and its derivatives certainly were not specific to MLL-rearranged ALL, we decided to further focus on this highly aggressive type of leukemia. Given that Irinotecan (the pro-drug of SN-38) has been increasingly used for the treatment of various pediatric solid tumors, we specifically chose this agent for further pre-clinical evaluation in pediatric MLL-rearranged ALL. Interestingly, shortly after engraftment, Irinotecan completely blocked leukemia expansion in mouse xenografts of a pediatric MLL-rearranged ALL cell line, as well as in two patient-derived xenograft (PDX) models of MLL-rearranged infant ALL. Also, from a more clinically relevant perspective, Irinotecan monotherapy was able to induce sustainable disease remissions in MLL-rearranged ALL xenotransplanted mice burdened with advanced leukemia. Taken together, our data demonstrate that Irinotecan exerts highly potent anti-leukemia effects against pediatric MLL-rearranged ALL, and likely against other, more favorable subtypes of childhood ALL as well.

Exploiting the reactive oxygen species imbalance in high-risk paediatric acute lymphoblastic leukaemia through auranofin.

The prognosis for high-risk childhood acute leukaemias remains dismal and established treatment protocols often cause long-term side effects in survivors. This study aims to identify more effective and safer therapeutics for these patients. A high-throughput phenotypic screen of a library of 3707 approved drugs and pharmacologically active compounds was performed to identify compounds with selective cytotoxicity against leukaemia cells followed by further preclinical evaluation in patient-derived xenograft models. Auranofin, an FDA-approved agent for the treatment of rheumatoid arthritis, was identified as exerting selective anti-cancer activity against leukaemia cells, including patient-derived xenograft cells from children with high-risk ALL, versus solid tumour and non-cancerous cells. It induced apoptosis in leukaemia cells by increasing reactive oxygen species (ROS) and potentiated the activity of the chemotherapeutic cytarabine against highly aggressive models of infant MLL-rearranged ALL by enhancing DNA damage accumulation. The enhanced sensitivity of leukaemia cells towards auranofin was associated with lower basal levels of the antioxidant glutathione and higher baseline ROS levels compared to solid tumour cells. Our study highlights auranofin as a well-tolerated drug candidate for high-risk paediatric leukaemias that warrants further preclinical investigation for application in high-risk paediatric and adult acute leukaemias.

Decitabine mildly attenuates MLL-rearranged acute lymphoblastic leukemia in vivo, and represents a poor chemo-sensitizer.

MLL‐rearranged acute lymphoblastic leukemia (ALL) represents a highly aggressive ALL subtype, characterized by aberrant DNA methylation patterns. DNA methyltransferase inhibitors, such as decitabine have previously been demonstrated to be effective in eradicating MLL‐rearranged ALL cells in vitro.Here, we assessed the in vivo anti‐leukemic potential of low‐dose DNA methyltransferase inhibitor decitabine using a xenograft mouse model of human MLL‐rearranged ALL. Furthermore, we explored whether prolonged exposure to low‐dose decitabine could chemo‐sensitize MLL‐rearranged ALL cells toward conventional chemotherapy as well as other known epigenetic‐based and anti‐neoplastic compounds.Our data reveal that decitabine prolonged survival in xenograft mice of MLL‐rearranged ALL by 8.5 days (P = .0181), but eventually was insufficient to prevent leukemia out‐growth, based on the examination of the MLLAF4 cell line SEM. Furthermore, we observe that prolonged pretreatment of low‐dose decitabine mildly sensitized toward the conventional drugs prednisolone, vincristine, daunorubicin, asparaginase, and cytarabine in a panel of MLL‐rearranged cell lines. Additionally, we assessed synergistic effects of decitabine with other epigenetic‐based or anticancer drugs using high‐throughput drug library screens. Validation of the top hits, including histone deacetylase inhibitor panobinostat, BCL2 inhibitor Venetoclax, MEK inhibitor pimasertib, and receptor tyrosine kinase foretinib, revealed additive and moderate synergistic effects for the combination of each drug together with decitabine in a cell line‐dependent manner.

Abstract 1745: Application of BET/CBP/p300 multi-bromodomain inhibitors as a novel therapeutic strategy for MLL-rearranged acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) is the most common form of childhood leukemia. While the prognosis of patients with ALL has improved significantly, ALL with chromosomal rearrangements involving mixed-lineage leukemia (MLL) gene remains an incurable disease. Thus, development of a safe and efficacious therapeutic agent based on the biology of MLL rearrangement is of high importance to address this unmet medical need. Bromodomains are protein motifs that bind to acetylated histones and are recognized as new epigenetic drug targets in recent years. Bromodomain and extra-terminal motif proteins (“BET proteins”) are epigenetic ‘reader' proteins that are characterized by containing two of such bromodomains. BRD4, a member of BET protein family, is a key component of multi-protein complex that induce oncogene transcription at Super-Enhancers. Currently, small-molecule BET inhibitors are being investigated in the clinic as potential therapeutics for hematological cancers. CBP and p300, two paralogous histone acetyltransferases, also contain bromodomains and are considered as drug targets for cancer. Because BET and CBP/p300 proteins are key components of the etiology of MLL-rearranged (MLL-r) leukemia, an agent that simultaneously target both classes of proteins may have therapeutic advantages. We previously demonstrated that a novel BET inhibitor, CG13250, suppressed multiple myeloma cell proliferation in vitro and in vivo (Biochem Biophys Res Commun, 484:262-268, 2017) and subsequently synthesized additional BET inhibitors (Bioorg Med Chem Lett, 29:1220-1226, 2019). In the present study, we demonstrate that some of our BET inhibitors can also inhibit bromodomains of CBP/p300 proteins. One such compound, CN470, potently suppresses BRD4, CBP, and p300 with BROMOscan KD values of 33, 32, and 20 nM, respectively, and suppressed mRNA and protein expression of c-MYC as expected. CN470 potently inhibited the growth of MLL-r ALL cell lines in a dose-dependent manner and flow cytometric analyses showed that CN470 caused apoptosis in MLL-r ALL cells following a cell cycle arrest at the G1 phase. Moreover, co-immunoprecipitation assay demonstrated that CN470 reduced the binding of BRD4 to acetylated H3 (H3K27), an effect absent with a reference BET inhibitor, OTX-015. Taken together, CN470 may represent an effective strategy against MLL-r ALL cells. As a confirmatory study, we investigated the in vivo effects of CN470 by using SEMLuc/GFP cells in an orthotopic mouse model of MLL-AF4 leukemia. Mice were orally administered with CN470 (10 mg/kg) once daily resulting in a prolonged survival compared to vehicle control group (mean 37 days vs. 28 days; p=0.0246). In conclusion, simultaneous inhibition of BET/CBP/p300 with a multi-bromodomain inhibitor represents a promising novel therapeutic approach against MLL-r ALL. Citation Format: Natsuki Imayoshi, Makoto Yoshioka, Shyh-Ming Yang, Koshi Akahane, Yuki Toda, Shigekuni Hosogi, Takeshi Inukai, Seiji Okada, David J. Maloney, Jefferey W. Strovel, Eishi Ashihara. Application of BET/CBP/p300 multi-bromodomain inhibitors as a novel therapeutic strategy for MLL-rearranged acute lymphoblastic leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1745.

MLL-Rearranged Acute Lymphoblastic Leukemia.

Rearrangements of the histone lysine [K]-MethylTransferase 2A gene (KMT2A) gene on chromosome 11q23, formerly known as the mixed-lineage leukemia (MLL) gene, are found in 10% and 5% of adult and children ALL cases, respectively. The most common translocated genes are AFF1 (formerly AF4), MLLT3 (formerly AF9), and MLLT1 (formerly ENL). The bimodal incidence of MLL-r-ALL usually peaks in infants in their first 2years of life and then declines thereafter during the pediatric/young adult phase until it increases again with age. MLL-rearranged ALL (MLL-r-ALL) is characterized by hyperleukocytosis, aggressive behavior with early relapse, relatively high incidence of central nervous system (CNS) involvement, and poor prognosis. MLL-r-ALL cells are characterized by relative resistance to corticosteroids (due to Src kinase-induced phosphorylation of annexin A2) and L-asparaginase therapy, but they are sensitive to cytarabine chemotherapy (due to increased levels of hENT1 expression). Potential therapeutic targets include FLT3 inhibitors, MEK inhibitors, HDAC inhibitors, BCL-2 inhibitors, MCL-1 inhibitors, proteasome inhibitors, hypomethylating agents, Dot1L inhibitors, and CDK inhibitors. In this review, we discuss MLL-r-ALL focusing on clinical presentation, risk stratification, drug resistance, and treatment strategies, including potential novel therapeutic targets.

A Menin-MLL Inhibitor Induces Specific Chromatin Changes and Eradicates Disease in Models of MLL-Rearranged Leukemia

Inhibition of the Menin (MEN1) and MLL (MLL1, KMT2A) interaction is a potential therapeutic strategy for MLL-rearranged (MLL-r) leukemia. Structure-based design yielded the potent, highly selective, and orally bioavailable small-molecule inhibitor VTP50469. Cell lines carrying MLL rearrangements were selectively responsive to VTP50469. VTP50469 displaced Menin from protein complexes and inhibited chromatin occupancy of MLL at select genes. Loss of MLL binding led to changes in gene expression, differentiation, and apoptosis. Patient-derived xenograft (PDX) models derived from patients with either MLL-r acute myeloid leukemia or MLL-r acute lymphoblastic leukemia (ALL) showed dramatic reductions of leukemia burden when treated with VTP50469. Multiple mice engrafted with MLL-r ALL remained disease free for more than 1 year after treatment. These data support rapid translation of this approach to clinical trials.

Targeting Critical Kinases and Anti-Apoptotic Molecules Overcomes Steroid Resistance in Infant MLL-Rearranged Leukemia

Acute lymphoblastic leukemia (ALL) with rearrangement of the mixed-lineage leukemia (MLL) gene frequently affects infants and is associated with a poor prognosis. Standard treatment protocol for infant MLL-rearranged ALL (MLL-ALL) includes glucocorticoids (GCs). However, resistance to GCs remains a major problem. Therefore, it is important to find new treatment strategies that overcome GC resistance in MLL-ALL. To identify novel therapeutic targets to overcome GC resistance in MLL-ALL, we compared transcriptional profiles of normal cord blood CD34+CD38- hematopoietic stem and progenitor cells and GC-resistant infant MLL-ALL leukemia-initiating cells (LICs). We found enrichment of the Src family kinases (SFKs) and Fms-like tyrosine kinase 3 (FLT3) signaling pathways in the LICs. We hypothesized that activation of these kinases may contribute to GC resistance in MLL-ALL cells. Using our previously developed infant MLL-ALL patient-derived xenograft (PDX) models (Aoki et al., Blood, 2015), we recapitulated human GC-resistance in vivo, characterized by increased hCD45+ chimerism in the peripheral blood after treatment with dexamethasone (% peripheral blood human MLL-ALL at pre-treatment: 34.1±5.2% vs. post treatment 51.3±6.6%, n=26). In order to investigate if inhibition of SFKs and FLT3 can overcome GC-resistance, we treated the MLL-ALL PDX models with our previously developed FLT3-SFK multiple kinase inhibitor RK-20449 (Saito et al., Science Translational Medicine, 2013). Combination treatment with dexamethasone and RK-20449 successfully eliminated human MLL-ALL cells from the peripheral blood (dexamethasone alone: 47.2±7.2% vs. combination: 5.6±2.8% hCD45+ cells, p=6.91E-7), bone marrow (dexamethasone alone: 72.2±4.6% vs. combination: 19.5±4.8% hCD45+ cells, p=1.79E-10), and spleen (dexamethasone alone: 52.6±4.8% vs. combination: 11.7±3.5% hCD45+ cells, p=3.75E-9). In addition, combination treatment eliminated infiltrated MLL-ALL from kidney and liver of the MLL-ALL PDX models. Bcl-2 homology domain 3 (BH3) profiling demonstrated that MLL-ALL cells resistant to RK-20449 treatment were dependent on the anti-apoptotic Bcl-2 protein for their survival. Additional inhibition of Bcl-2 by ABT-199 led to complete elimination of MLL-ALL cells in vitro and in vivo in all MLL-ALL patient cases (Figure1). Taken together, in this study we demonstrated that inhibition of SFKs and FLT3 by RK-20449 overcomes GC-resistance in MLL-ALL. Further, we identified Bcl-2 dependence as a mechanism of treatment resistance in MLL-ALL. Therefore, we believe that the combined inhibition of kinase and anti-apoptotic pathways may lead to effective treatment options for highly resistant infant MLL-ALL. Aoki, et al., Identification of CD34+ and CD34- leukemia-initiating cells in MLL-rearranged human acute lymphoblastic leukemia. Blood, 2015: p. 967-980. Saito, Y., et al., A Pyrrolo-Pyrimidine Derivative Targets Human Primary AML Stem Cells in Vivo. Science Translational Medicine, 2013: p. 1-15. Disclosures No relevant conflicts of interest to declare.

V2 Trial: A Phase I Study of Venetoclax Combined with CPX-351 for Children, Adolescents and Young Adults with Relapsed or Refractory Acute Leukemia

Despite significant advances in AML therapy over the past decades, 30-40% of all young patients with AML will suffer a relapse, after which achieving long-term disease-free survival remains challenging. In pediatric and young adult patients with AML, curative-intent treatment at relapse involves intensive induction therapy to achieve CR followed by consolidation with HSCT. Historically, initial response rates to salvage therapy were limited. Recently, Children's Oncology Group presented results from a Phase II study of CPX-351 (liposomal cytarabine:daunorubicin at a synergistic ratio) in pediatric patients with AML in first relapse demonstrating an impressive CR/CRp/CRi rate of 81.3%. Separately, we presented data from our first-in-pediatrics CPX-351 Phase I trial showing 48% M1 marrow response in a population consisting of children with multiply relapsed and refractory (R/R) AML patients. We also obtained single cell RNA sequencing on study patients' peripheral blasts before, during, and after CPX-351 treatment (i.e.: Days 0, 1 and 5). Preliminary data showed that CPX-351 differentially impacts p53 targets within leukemic blast clusters over time, with an enrichment for genes regulating apoptosis (e.g.: FAS, BAX) in most clusters. This suggests that these blasts may be primed for apoptosis following CPX-351 treatment. Anti-apoptosis signaling in leukemias is an established mechanism of chemoresistance. Venetoclax is an orally available, small molecule inhibitor of anti-apoptotic protein BCL-2, which is frequently overexpressed in leukemias. Based on published preclinical data and single cell RNA sequencing data from our CPX-351 trial, we developed a Phase I study to investigate the combination of CPX-351 and venetoclax in the treatment of young patients with R/R acute leukemias. The V2 trial is a single-institution Phase I pilot study (NCT03826992) designed to test the safety and tolerability of combining venetoclax with CPX-351 in patients ages 1-39 years for the treatment of R/R acute leukemias. Inclusion diagnoses are based on preclinical sensitivities and include AML, MPAL, or AUL in first or greater relapse, or ETP-ALL, MLL-rearranged ALL, or T-ALL in second or greater relapse. Patients must have measurable disease. Exclusion criteria include CNS status 3, bone marrow failure syndromes, and prior anthracycline and mediastinal radiation exposures above acceptable cardiotoxicity risk thresholds. Subjects receive a single course of study therapy consisting of daily venetoclax and CPX-351 at the FDA approved dose for adults on Days 1, 3, and 5 (Fig. 1) with the goal of CRMRD- to allow for off-study HSCT. The venetoclax dose exploration phase begins with a starting dose of 400 mg (or the allometrically-scaled equivalent) for 21 days with a 3-day ramp to target dose. The dose exploration phase proceeds on a rolling 6 design and will require 4-12 patients to determine RP2D. Provided a tolerable RP2D is found, a dose expansion phase will open. Primary endpoints are determination of RP2D and description of the safety profile including time to hematologic recovery. Secondary endpoints are estimations of CR/CRp/CRi +/- MRD- within the context of a phase I study, and evaluation of cancer therapy-related cardiac dysfunction (CTRCD) rates. Correlative studies include evaluation of venetoclax PK with concomitant CPX-351 using modeling &amp; simulation with a combined limited sampling and pediatric opportunistic PK sampling (POPS) strategy, and exploration of the correlations between BCL-2 family expression and pediatric tumor genetics with treatment response. Figure 1 Disclosures O'Brien: BTG: Research Funding; Pfizer: Research Funding; Celgene: Research Funding; AbbVie: Research Funding; Amgen: Research Funding; BMS: Research Funding. Phillips:Novartis: Membership on an entity's Board of Directors or advisory committees. Vinks:Myriad Genetics: Consultancy, Patents &amp; Royalties. Perentesis:Kurome Therapeutics: Consultancy. Absalon:Jazz Pharmaceuticals: Other: CPX-351 for clinical trial . OffLabel Disclosure: This clinical trial involves the off-label use of CPX-351 and venetoclax for relapsed/refractory pediatric and young adult leukemias.