Disease relapse in patients with acute myeloid leukemia (AML) is associated with a failure of current treatments to eradicate leukemia stem cells (LSCs), a self-renewing population of cells responsible for disease progression and maintenance. Thus, novel therapeutic strategies designed to specifically target LSCs while sparing normal hematopoietic stem cells are needed. To identify dependencies in LSCs that may reveal new treatment opportunities, we performed an in vivo CRISPR/Cas9 dropout screen in the widely used MLL-AF9-driven AML murine model. The pooled lentiviral CRISPR library was designed to target 960 genes encoding cell surface proteins expressed on MLL-AF9 AML cells as these are accessible for therapeutic targeting. The facilitated glucose transporter member 1(GLUT1), a major mediator of cellular glucose uptake, emerged as the highest ranked dependency in the screen, with all 6 sgRNAs depleted more than 10-fold in vivo. Consistent with the results from the screen, validation experiments confirmed that sgRNA-mediated GLUT1 disruption in c-Kit +Cas9 +dsRed +MLL-AF9 cells led to a 5-fold reduction in the establishment of leukemia in both the bone marrow and spleen of recipient mice. In line with these in vivo observations, leukemia cells expressing GLUT1 sgRNAs were rapidly depleted over time in an ex vivo competition assay (p<0.0001). GLUT1 disruption also led to a marked increase in mean survival from 28 to 73 days in mice transplanted with sorted GLUT1 sgRNA-expressing leukemia cells relative to controls. Notably, while GLUT1 loss did not affect apoptosis or cell-cycle state, it led to a more than two-fold increase in the surface expression of the myeloid differentiation marker Gr-1 (p=0.0002). Interestingly, knockdown of GLUT1 lead to reduced mRNA expression levels of key downstream genes of MLL-driven leukemia Meis1 (p<0.0001) and Hoxa9 (p=0.0013) , both of which are commonly downregulated upon differentiation. These findings suggest that GLUT1 ablation arrests AML cell growth at least in part via accelerated differentiation and attenuated cell proliferation. Given GLUT1-mediated glucose transfer constitutes the first rate-limiting step for glucose metabolism, we assessed the metabolic profile of MLL-AF9 AML cells following loss of GLUT1. Bioenergetic profiling revealed that the rate of glycolysis was significantly decreased upon GLUT1 knockdown, as measured by a decrease in extracellular acidification rate (ECAR), glucose uptake, hexokinase activity and extracellular lactate production. To further assess the feasibility of GLUT1 inhibition as a therapy for AML patients, we treated murine cKit +MLL-AF9 leukemia cells with BAY-876, a potent and highly selective GLUT1 inhibitor. BAY-876 impaired tumor growth following 24hr (IC 50 60.3 nM) and 48hr (IC 50 68.8 nM) treatment ex vivo in a dose-dependent manner. Interestingly, the inhibitory effect on the counterpart healthy bone marrow c-Kit + cells was significantly weaker (24hr IC 50 347.7 nM; 48hr IC 50 258.4nM), indicating selective targeting of LSCs. To test the efficacy of BAY-876 as an anti-leukemic agent in vivo, sublethally irradiated mice were transplanted with c-Kit +MLL-AF9 AML cells and 3 days post-injection, were randomised into two groups (Veh n=4; BAY-876 n=6) and orally treated with either vehicle or 4mg/kg of BAY-876 daily. Following 10 days of treatment, mice were sacrificed and leukemia burden was assessed. Notably, substantially lower levels of leukemia cells in the bone marrow (p=0.0095), spleen (p=0.0095), and peripheral blood (p=0.036) were observed in the BAY-876 treatment group with no significant loss of body weight. Consistent with these findings, the average spleen weight was reduced by 66% upon BAY-876 treatment (p=0.0136). Collectively, we demonstrate that MLL-AF9-driven AML cells are dependent on GLUT1 for continued growth and survival. Targeting of GLUT1 downregulates glycolysis and induces cellular differentiation. We report that genetic or pharmacological inhibition of GLUT1 is sufficient to impair leukemic growth in vitro and in vivo, highlighting a potential therapeutic opportunity for disarming intrinsic metabolic dependencies of LSCs. Ongoing studies are aimed at translating these findings to the human disease and exploring combinatorial therapies that may act synergistically to overcome mechanisms of therapy resistance and metabolic plasticity. Disclosures No relevant conflicts of interest to declare.
Genetic rearrangements involving the KMT2A gene (KMT2A-R) are seen in around 10% of acute leukemia overall. KMT2A-R occurs in all ages and usually correlates with high-risk clinical features, in particular in infants aged 0-12 months of age with acute lymphoblastic leukemia (ALL). To uncover age- and leukemia-subtype specific molecular patterns in KMT2A-R ALL and acute myeloid leukemia (AML), we performed whole genome (WGS), whole exome (WES), and RNA-sequencing on a well-annotated Nordic KMT2A-R cohort of 104 patients, including infant ALL (n=33), childhood ALL (n=18), adult ALL (n=15), and pediatric AML (n=38) patients. For 77 patients, we performed WGS (40x) at diagnosis and remission as well as WES (140x) on the diagnostic sample, and remaining patients underwent WES only (n=27). RNA-sequencing was performed on 58 cases with available RNA. Twenty-two genes were recurrently altered and remarkably, NRAS, KRAS, FLT3, PAX5, TP53, CDKN2A/B and IKZF1 accounted for 70% of mutations. The landscape of mutations suggested the presence of leukemia and age-specific associations with MYST4, PTPN11, and SETD2 uniquely altered in AML and PIK3CD, DNAH11, NOTCH1, CSMD3 and CDKN2A/B in ALL. Some genes were mutated in both KMT2A-R ALL and AML, but were more common in one disease, such as FLT3 and KRAS in AML and PAX5, TP53 and IKZF1 in ALL. Moreover, age-associated patterns were seen in ALL with NRAS more frequently mutated than KRAS in infant ALL (26% vs 15%), and KRAS more frequently mutated than NRAS in childhood ALL (24% vs 18%), with adult ALL having fewer such mutations (NRAS 13%; KRAS 7%). Alterations of CDKN2A/B and TP53 were absent in infant ALL, detected in childhood and adult ALL only. PAX5 alterations were primarily detected in childhood ALL (22%, 9% infant ALL, 7% adult ALL), with all three PAX5-altered infant cases having the KMT2A-MLLT3 fusion gene. Finally, KMT2A-R pediatric AML had the highest fraction of FLT3 mutations (24%, 9% infant ALL, 11% childhood ALL, 0% adult ALL) and all but one mutation occurred in KMT2A-MLLT3 rearranged cases and most were kinase domain point mutations. We next expanded our analysis to include non-recurrent alterations. PI3K/RAS pathway alterations were detected across ages and subtypes with the highest fraction in pediatric AML (63%) and the lowest in adult ALL (27%, 43% infant ALL, 41% childhood ALL). Further, cell cycle related genes were primarily mutated in childhood (39%) and adult ALL cases (33%) and rarer in infant ALL (12%) and pediatric AML (16%) and genes within the B-cell pathway were more commonly altered in childhood ALL (29%) than in infant ALL (9%). Finally, in line with our previous study (Andersson et al, Nat Genet 2015) epigenetic mutations were absent in infant ALL, but present in 20-35% of the other patients. RNA-sequencing identified the KMT2A-fusion in 56/58 cases, with low exonic coverage preventing detection of the fusion in two cases. The reciprocal KMT2A fusion was only expressed in 13/39 cases where it was predicted to be expressed based on karyotype or whole genome sequencing data with 11/13 cases having the KMT2A-AFF1 fusion gene. In addition, RNA-sequencing identified 6 in-frame and 12 out-of-frame fusion genes that had formed either as part of the KMT2A-R itself or that were independent genetic events. Further, a novel in-frame KMT2A-ACIN1 fusion was identified in a child aged 1 year with B-precursor ALL. ACIN1 encodes Apoptotic Chromatin Condensation Inducer 1 and the fusion was formed through an insertion of 14q11 into 11q23. ACIN1 is also rearranged as part of the ACIN1-NUTM1 that we identified in an infant with ins(15;14)(q22;q11.2q32.1) (Andersson et al Nat Genet 2015). To study the ability of KMT2A-ACIN1 to induce leukemia in mice, we injected retrovirally transduced mouse bone marrow cells containing the fusion into syngeneic mice and KMT2A-MLLT3 was used as control. All mice succumbed to disease at an average of 112 days for KMT2A-ACIN1 (n=12) and 63 days for KMT2A-MLLT3 (n=5) and mice displayed splenomegaly and leukocytosis with an immunophenotype indicative of AML. Primary leukemia cells isolated from moribund mice gave rise to leukemia in sublethally irradiated recipients with reduced disease latency. In conclusion, these results highlight the differential molecular patterns in KMT2A-R leukemia across infancy to adulthood thereby providing novel pathogenetic insight. Disclosures No relevant conflicts of interest to declare.
Targeted therapies exploiting vulnerabilities of cancer cells hold promise for improving patient outcome and reducing side effects of chemotherapy. Yet, mechanistic understanding linking drug effects and cancer cell state diversity is crucial for identifying effective combination therapies to prevent disease recurrence. Here, we characterized at the level of gene regulatory networks the effect of G2/M cell cycle checkpoint inhibition in acute lymphoblastic leukemia (ALL) and demonstrate that WEE1 targeted therapy impinges on cell fate regulatory circuits. We found highest inhibition of recovery of proliferation in KMT2A-rearranged (KMT2A-r) ALL cells, compared to other leukemia subgroups. Single-cell transcriptome and chromatin accessibility profiling of (KMT2A::AFF1) RS4;11 cells treated with AZD1775 revealed strong activation of p53-driven processes linked to induction of apoptosis and senescence, and disruption of a core KMT2A-RUNX1-MYC regulatory network through CDK1-mediated RUNX1 degradation. In RS4;11 cells and in patient-derived xenograft (PDX) model, we uncovered that a transition to a cell state characterized by activation of transcription factors regulating pre-B cell fate, lipid metabolism and pre-BCR signaling supported a drug tolerance. By sequential treatment targeting the drug tolerant cell state phenotype with BCR-signaling inhibitors dasatinib, ibrutinib, or perturbing metabolism by fatostatin or AZD2014 after AZD1775 administration, this cell state evolution underlying recovery of leukemic cells could be blocked. Collectively, our findings provide new insights into the tight connectivity of gene regulatory programs associated with cell cycle and cell fate regulation, and a rationale for sequential administration of WEE1 inhibitors with low toxicity inhibitors of pre-BCR signaling or metabolism.
Our understanding of how individual mutations, whether present in all or just a fraction of the leukemia cells, affect cellular responses to therapy is limited. Leukemia mouse models provide a unique possibility to explore how therapy affects the evolution of genetically distinct clones and identify mechanisms of resistance allowing transfer to human disease. Herein, we studied how different therapies influenced survival, clonal evolution, and resistance patterns in mouse KMT2A-MLLT3 leukemia with subclonal FLT3 N676K. Bone marrow (BM) from a leukemia expressing KMT2A-MLLT3-mCherry in all cells and a FLT3 N676K-GFP in 40% of cells, were re-transplanted to sublethally irradiated recipients (Hyrenius-Wittsten el al, Nat Commun, 2018). Upon engraftment, treatment was started with either chemotherapy (cytarabine for 5 days + doxorubicin for 3 days), the FLT3 inhibitor AC220, chemotherapy followed by AC220, or AC220+Trametinib, a MEK inhibitor. Targeted treatment was given for 28 days; controls received vehicle (Fig. 1a). Survival was estimated by Kaplan-Meier and the developing leukemias were analyzed by flow-cytometry, RNA-sequencing and targeted gene re-sequencing. Each treatment prolonged survival with a median latency of 30 days for chemotherapy , 37.5 days for AC220, 42 days for chemotheraphy+AC220, and 45 days for AC220+Trametenib, versus 25.5 days for the control (Fig. 1b). Most leukemia cells expressed GFP/mCherry and mice displayed splenomegaly and leukocytosis. Next, we investigate how treatment impacted evolution of the KMT2A-MLLT3+FLT3 N676K cells and while they constituted all cells in control and chemotherapy-treated mice, the other treatments impacted their evolution. Three distinct patterns were discerned with either >80% of KMT2A-MLLT3+FLT3 N676K cells, >80% of cells expressing KMT2A-MLLT3 alone, or dual similar sized clones of cells expressing KMT2A-MLLT3 alone or KMT2A-MLLT3+FLT3 N676K(Fig. 1c). Eradication of the FLT3-leukemia cells was rare, but most common in mice receiving AC220+Trametinib and the frequency of dual clones increased when mice received chemotherapy followed by AC220, in line with treatment selectively affecting evolution of genetically distinct cells (Fig. 1d). To find clues to treatment resistance, RNA-sequencing (N=44) revealed segregation into three major clusters: 1) leukemias expressing KMT2A-MLLT3 alone, 2) control and chemotherapy-treated leukemias and 3) AC220 treated leukemias. Notably, a set of AC220-treated mice clustered close to the control and chemotherapy-treated mice (Fig. 1e). Flow-cytometry data showed that similar to the control and chemotherapy-treated leukemias, the myeloid BM cells of those AC220 samples, aberrantly expressed B220 (Fig. 1f). Gene set enrichment analysis revealed enrichment of gene sets correlating with stem cells and oxidative phosphorylation in those AC220-treated leukemias, suggesting a switch in cellular phenotype and metabolic state upon treatment. By contrast, the other AC220 leukemias (cluster 3), instead showed enrichment of gene sets correlating with granulocyte/macrophage progenitors and immune regulatory pathways, indicating selective dependence of distinct cellular pathways upon resistance (Fig. 1g). Finally, acquisition of AC220 resistance mutations was rare with a FLT3 D835Y and a Ptpn11 G503V detected in two leukemias only. Taken together, these results show that the specific treatment given not only affected survival of the FLT3 N676K mutated KMT2A-MLLT3 leukemia, but also impacted how the genetically distinct cells evolved. The general lack of acquired mutations upon targeted treatment suggests that target-independent mechanisms that result in alternate activation of survival/proliferation explains acquired resistance in a majority of mice and provides novel insights into treatment resistance. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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