Identification of Genes Transcriptionally Responsive to the Loss of MLL Fusions in MLL-Rearranged Acute Lymphoblastic Leukemia

Introduction

MLL-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year) is characterized by high relapse rates and a dismal prognosis. To facilitate the discovery of novel therapeutic targets, we here searched for genes directly influenced by the repression of various MLL fusions.

Methods

For this, we performed gene expression profiling after siRNA-mediated repression of MLL-AF4, MLL-ENL, and AF4-MLL in MLL-rearranged ALL cell line models. The obtained results were compared with various already established gene signatures including those consisting of known MLL-AF4 target genes, or those associated with primary MLL-rearranged infant ALL samples.

Results

Genes that were down-regulated in response to the repression of MLL-AF4 and MLL-ENL appeared characteristically expressed in primary MLL-rearranged infant ALL samples, and often represented known MLL-AF4 targets genes. Genes that were up-regulated in response to the repression of MLL-AF4 and MLL-ENL often represented genes typically silenced by promoter hypermethylation in MLL-rearranged infant ALL. Genes that were affected in response to the repression of AF4-MLL showed significant enrichment in gene expression profiles associated with AF4-MLL expressing t(4;11)+ infant ALL patient samples.

Conclusion

We conclude that the here identified genes readily responsive to the loss of MLL fusion expression potentially represent attractive therapeutic targets and may provide additional insights in MLL-rearranged acute leukemias.     

Internal Tandem Duplication Mutations in FLT3 Gene Augment Chemotaxis to Cxcl12 Protein by Blocking the Down-regulation of the Rho-associated Kinase via the Cxcl12/Cxcr4 Signaling Axis

Internal tandem duplication mutations in the Flt3 gene (ITD-FLT3) enhance cell migration toward the chemokine Cxcl12, which is highly expressed in the therapy-protective bone marrow niche, providing a potential mechanism underlying the poor prognosis of ITD-FLT3⁺ acute myeloid leukemia. We aimed to investigate the mechanisms linking ITD-FLT3 to increased cell migration toward Cxcl12. Classification of the expression of Cxcl12-regulated genes in ITD-FLT3⁺ cells demonstrated that the enhanced migration of ITD-FLT3⁺ cells toward Cxcl12 was associated with the differential expression of genes downstream of Cxcl12/Cxcr4, which are functionally distinct from those expressed in ITD-FLT3⁻ cells but are independent of the Cxcr4 expression levels. Among these differentially regulated genes, the expression of Rock1 in the ITD-FLT3⁺ cells that migrated toward Cxcl12 was significantly higher than in ITD-FLT3⁻ cells that migrated toward Cxcl12. In ITD-FLT3⁻ cells, Rock1 expression and Mypt1 phosphorylation were transiently up-regulated but were subsequently down-regulated by Cxcl12. In contrast, the presence of ITD-FLT3 blocked the Cxcl12-induced down-regulation of Rock1 and early Mypt1 dephosphorylation. Likewise, the FLT3 ligand counteracted the Cxcl12-induced down-regulation of Rock1 in ITD-FLT3⁻ cells, which coincided with enhanced cell migration toward Cxcl12. Rock1 antagonists or Rock1 shRNA abolished the enhanced migration of ITD-FLT3⁺ cells toward Cxcl12. Our findings demonstrate that ITD-FLT3 increases cell migration toward Cxcl12 by antagonizing the down-regulation of Rock1 expression. These findings suggest that the aberrant modulation of Rock1 expression and activity induced by ITD-FLT3 may enhance acute myeloid leukemia cell chemotaxis to the therapy-protective bone marrow niche, where Cxcl12 is abundantly expressed.     

Inhibition of FLT3 Expression by Green Tea Catechins in FLT3 Mutated-AML Cells

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by a block in differentiation and uncontrolled proliferation. FLT3 is a commonly mutated gene found in AML patients. In clinical trials, the presence of a FLT3-ITD mutation significantly correlates with an increased risk of relapse and dismal overall survival. Therefore, activated FLT3 is a promising molecular target for AML therapies. In this study, we have shown that green tea polyphenols including (−)-epigallocatechin-3-gallate (EGCG), (−)-epigallocatechin (EGC), and (−)-epicatechin-3-gallate (ECG) suppress the proliferation of AML cells. Interestingly, EGCG, EGC and ECG showed the inhibition of FLT3 expression in cell lines harboring FLT3 mutations. In the THP-1 cells harboring FLT3 wild-type, EGCG showed the suppression of cell proliferation but did not suppress the expression of FLT3 even at the concentration that suppress 100% cell proliferation. Moreover, EGCG-, EGC-and ECG-treated cells showed the suppression of MAPK, AKT and STAT5 phosphorylation. Altogether, we suggest that green tea polyphenols could serve as reagents for treatment or prevention of leukemia harboring FLT3 mutations.     

Expression of the FLT3-ITD oncogene sensitizes murine progenitor B-cell line BAF3 to cytotoxic action of binase

Acute myeloid leukemia makes up about 30% of all leukemia cases in adults. Mutations in the genes of the receptor tyrosine kinases KIT and FLT3, along with chromosomal translocations, are frequently found in leukemic cells. In the current work, we show that the transgenic B-cells BAF3/FLT3-ITD are significantly more sensitive to cytotoxic action of the ribonuclease binase than original BAF3 cells. BAF3/FLT3-ITD cells differ from BAF3 in expression of the FLT3-ITD oncogene, which results in the alteration of normal signaling pathways. We observed a similar effect previously when studying binase cytotoxic action in cells Kasumi-1 and FDC-P1-N822K, in which the activated oncogene KIT-N822K was expressed. An elevated cytotoxicity of binase to the cells that express the FLT3-ITD oncogene indicates that, as in case of the FDC-P1 cells transduced by the KIT oncogene, the expression of an activated oncogene determines the cell’s sensitivity to the binase action.     

The Novel Phospholipid Mimetic KPC34 Is Highly Active Against Acute Myeloid Leukemia with Activated Protein Kinase C

Acute myeloid leukemia (AML) is an aggressive malignancy with poor outcomes. Nucleoside analogs are subject to resistance mechanisms including downregulation of equilibrative nucleoside transporter (ENT1) and deoxycytidine kinase (dCK). KPC34 is a novel phospholipid mimetic that when cleaved by phospholipase C (PLC) liberates gemcitabine monophosphate and a diacylglycerol mimetic that inhibits the classical isoforms of protein kinase C (PKC). KPC34 acts independently of ENT1 and dCK. KPC34 was active against all AML cell lines tested with IC₅₀s in the nanomolar range. Enforced expression of PLC increased response to KPC34 in vivo. In an orthotopic, xenograft model, KPC34 treatment resulted in a significant increase in survival compared to control animals and those treated with high-dose cytarabine. In a PDX model with activated PKC, there was a significant survival benefit with KPC34, and at progression, there was attenuation of PKC activation in the resistant cells. In contrast, KPC34 was ineffective against a syngeneic, orthotopic AML model without activated PKC. However, when cells from that model were forced to express PKC, there were significantly increased sensitivity in vitro and survival benefit in vivo. These data suggest that KPC34 is active against AML and that the presence of activated PKC can be a predictive biomarker.     

Leukemic transformation of hematopoietic cells in mice internally exposed to depleted uranium

Depleted uranium (DU) is a dense heavy metal used in military applications. During military conflicts, US military personnel have been wounded by DU shrapnel. The health effects of embedded DU are unknown. Published data from our laboratory demonstrated that DU exposure in vitro can transform immortalized human osteoblast cells (HOS) to the tumorigenic phenotype. Results from our laboratory have also shown that DU is genotoxic and mutagenic in cultured human cells. Internalized DU could be a carcinogenic risk and concurrent alpha particle and heavy metal toxic effects complicate this potential risk. Anecdotal reports have suggested that DU can cause leukemia. To better assess this risk, we have developed an in vivo leukemogenesis model. This model involves using murine hematopoietic cells (FDC-P1) that are dependent on stimulation by granulocyte-macrophage colony stimulating factor (GM-CSF) or interleukin 3 (IL-3) and injected into mice to produce myeloid leukemia. Although immortalized, these cells are not tumorigenic on subcutaneous inoculation in mice. Intravenous injection of FDC-P1 cells into DU-implanted DBA/2 mice was followed by the development of leukemias in 76% of all mice implanted with DU pellets. In contrast, only 12% of control mice developed leukemia. Karyotypic analysis confirmed that the leukemias originated from FDC-P1 cells. The growth properties of leukemic cells from bone marrow, spleen, and lymph node were assessed and indicate that the FDC-P1 cells had become transformed in vivo. The kidney, spleen, bone marrow, muscle, and urine showed significant elevations in tissue uranium levels prior to induction of leukemia. These results demonstrated that a DU altered in vivo environment may be involved in the pathogenesis of DU induced leukemia in an animal model.     

GZD824 as a FLT3, FGFR1 and PDGFRα Inhibitor Against Leukemia In Vitro and In Vivo

GZD824 is a novel third-generation BCR-ABL inhibitor. It entered Phase II clinical trials in China and Phase Ib clinical trials in USA in 2019 for treatment of patients with resistant chronic myeloid leukemia (CML). We found that at concentrations below 10 nM, GZD824 significantly suppresses FLT3, FGFR1 and PDGFRα kinase activities and inhibits their signal pathways in MV4-11^Flt3-ITD, KG-1^{FGFR1OP2-FGFR1} and EOL-1^{FIP1L1-PDGFRa} leukemia cells. It selectively inhibits the growth of MV4-11^Flt3-ITD, KG-1^{FGFR1OP2-FGFR1} and EOL-1^{FIP1L1-PDGFRa} cells, and also effectively suppresses the growth of Ba/F3-FLT3-ITD cells harboring F691I and other mutations with IC₅₀ values <10 nM. GZD824 induces G0/G1 phase arrest and apoptosis in MV4-11, KG-1 and EOL-1 cells and activates cleavage of caspase-3 and PARP. In MV4-11, Ba/F3-ITD-F691I and KG-1 mouse xenograft models, GZD824 at 10 or 20 mg/kg, q2d, p.o. almost completely eradicates tumors. It also inhibits the viability of primary leukemic blasts from a FLT3-ITD positive AML patient but not those expressing native FLT3. Thus GZD824 suppresses leukemia cells of FLT3-ITD-driven AML and other hematologic malignancies driven by FGFR1 or PDGFRa, and it may be considered to be a novel agent for the treatment of leukemia.     

Selective Akt Inhibitors Synergize with Tyrosine Kinase Inhibitors and Effectively Override Stroma-Associated Cytoprotection of Mutant FLT3-Positive AML Cells

Objectives

Tyrosine kinase inhibitor (TKI)-treated acute myeloid leukemia (AML) patients commonly show rapid and significant peripheral blood blast cell reduction, however a marginal decrease in bone marrow blasts. This suggests a protective environment and highlights the demand for a better understanding of stromal:leukemia cell communication. As a strategy to improve clinical efficacy, we searched for novel agents capable of potentiating the stroma-diminished effects of TKI treatment of mutant FLT3-expressing cells.

Methods

We designed a combinatorial high throughput drug screen using well-characterized kinase inhibitor-focused libraries to identify novel kinase inhibitors capable of overriding stromal-mediated resistance to TKIs, such as PKC412 and AC220. Standard liquid culture proliferation assays, cell cycle and apoptosis analysis, and immunoblotting were carried out with cell lines or primary AML to validate putative candidates from the screen and characterize the mechanism(s) underlying observed synergy.

Results and Conclusions

Our study led to the observation of synergy between selective Akt inhibitors and FLT3 inhibitors against mutant FLT3-positive AML in either the absence or presence of stroma. Our findings are consistent with evidence that Akt activation is characteristic of mutant FLT3-transformed cells, as well as observed residual Akt activity following FLT3 inhibitor treatment. In conclusion, our study highlights the potential importance of Akt as a signaling factor in leukemia survival, and supports the use of the co-culture chemical screen to identify agents able to potentiate TKI anti-leukemia activity in a cytoprotective microenvironment.     

Use of Genome Engineering to Create Patient Specific MLL Translocations in Primary Human Hematopoietic Stem and Progenitor Cells

One of the challenging questions in cancer biology is how a normal cell transforms into a cancer cell. There is strong evidence that specific chromosomal translocations are a key element in this transformation process. Our studies focus on understanding the developmental mechanism by which a normal stem or progenitor cell transforms into leukemia. Here we used engineered nucleases to induce simultaneous specific double strand breaks in the MLL gene and two different known translocation partners (AF4 and AF9), which resulted in specific chromosomal translocations in K562 cells as well as primary hematopoietic stem and progenitor cells (HSPCs). The initiation of a specific MLL translocation in a small number of HSPCs likely mimics the leukemia-initiating event that occurs in patients. In our studies, the creation of specific MLL translocations in CD34+ cells was not sufficient to transform cells in vitro. Rather, a variety of fates was observed for translocation positive cells including cell loss over time, a transient proliferative advantage followed by loss of the clone, or a persistent proliferative advantage. These studies highlight the application of genome engineering tools in primary human HSPCs to induce and prospectively study the consequences of initiating translocation events in leukemia pathogenesis.     

H2O2 production downstream of FLT3 is mediated by p22phox in the endoplasmic reticulum and is required for STAT5 signalling

The internal tandem duplication (ITD) of the juxtamembrane region of the FLT3 receptor has been associated with increased reactive oxygen species (ROS) generation in acute myeloid leukemia (AML). How this elevated level of ROS contributes to the leukemic phenotype, however, remains poorly understood. In this work we show that ROS in the FLT3-ITD expressing AML cell line MV4-11 is reduced by treatment with PKC412, an inhibitor of FLT3, DPI, a flavoprotein inhibitor, and VAS2870, a Nox specific inhibitor, suggesting that ROS production is both FLT3 and NADPH oxidase dependent. The majority of these ROS co-localize to the endoplasmic reticulum (ER), as determined with the H(2)O(2)-specific aryl-boronate dye Peroxyorange 1, which also corresponds to co-localization of p22phox. Moreover, knocking down p22phox dramatically reduces H(2)O(2) after 24 hours in the ER, without affecting mitochondrial ROS. Significantly, the FLT3 inhibitor PKC412 reduces H(2)O(2) in FLT3-ITD expressing cell lines (MV4-11, MOLM-13) through reduction of p22phox over 24 hours. Reduced p22phox is achieved by proteasomal degradation and is prevented upon GSK3-β inhibition. Knockdown of p22phox resulted in reduced STAT5 signalling and reduced Pim-1 levels in the cells after 24 hours. Thus, we have shown that FLT3 driven H(2)O(2) production in AML cells is mediated by p22phox and is critical for STAT5 signalling.     

Human Flt3 is expressed at the hematopoietic stem cell and the granulocyte/macrophage progenitor stages to maintain cell survival

FLT3/FLK2, a member of the receptor tyrosine kinase family, plays a critical role in maintenance of hematopoietic homeostasis, and the constitutively active form of the FLT3 mutation is one of the most common genetic abnormalities in acute myelogenous leukemia. In murine hematopoiesis, Flt3 is not expressed in self-renewing hematopoietic stem cells, but its expression is restricted to the multipotent and the lymphoid progenitor stages at which cells are incapable of self-renewal. We extensively analyzed the expression of Flt3 in human (h) hematopoiesis. Strikingly, in both the bone marrow and the cord blood, the human hematopoietic stem cell population capable of long-term reconstitution in xenogeneic hosts uniformly expressed Flt3. Furthermore, human Flt3 is expressed not only in early lymphoid progenitors, but also in progenitors continuously along the granulocyte/macrophage pathway, including the common myeloid progenitor and the granulocyte/macrophage progenitor. We further found that human Flt3 signaling prevents stem and progenitors from spontaneous apoptotic cell death at least through up-regulating Mcl-1, an indispensable survival factor for hematopoiesis. Thus, the distribution of Flt3 expression is considerably different in human and mouse hematopoiesis, and human FLT3 signaling might play an important role in cell survival, especially at stem and progenitor cells that are critical cellular targets for acute myelogenous leukemia transformation.     

Discovery of quinolinone derivatives as potent FLT3 inhibitors

Recently some fms-like tyrosine kinase 3 (FLT3) inhibitors have shown good efficacy in acute myeloid leukemia (AML) patients. In an effort to develop anti-leukemic drugs, we investigated quinolinone derivatives as novel FLT3 inhibitors. Two substituted quinolinones, KR65367 and KR65370 were subjected to FLT3 kinase activity assay and showed potent inhibition against FLT3 kinase activity in vitro, with IC₅₀ of 2.7 and 0.57 nM, respectively. As a measure of selectivity, effects on the activity of other kinases were also tested. Both compounds have negligible activity against Met, Ron, epidermal growth factor receptor, Aurora A, Janus kinase 2, and insulin receptor; with IC₅₀ greater than 10 μM. KR compounds showed strong growth inhibition in MV4;11 AML cells and increased the apoptotic cell death in flow cytometric analyses. A decrease in STAT5 phosphorylation by KR compounds was observed in MV4;11 cells. Furthermore, in vitro evaluation of compounds structurally related to KR65367 and KR65370 showed a good structure-activity relationship.     

PTEN is indispensable for cells to respond to MAPK inhibitors in myeloid leukemia

Constitutively activated MAPK and AKT signaling pathways are often found in solid tumors and leukemias. PTEN is one of the tumor suppressors that are frequently found deficient in patients with late-stage cancers or leukemias. In this study we demonstrate that a MAPK inhibitor, PD98059, inhibits both AKT and ERK phosphorylation in a human myeloid leukemia cell line (TF-1), but not in PTEN-deficient leukemia cells (TF-1a). Ectopic expression of wild-type PTEN in myeloid leukemia cells restored cytokine responsiveness at physiological concentrations of GM-CSF (<0.02 ng/mL) and significantly improved cell sensitivity to MAPK inhibitor. We also found that Early Growth Response 1 (EGR1) was constitutively over-expressed in cytokine-independent TF-1a cells, and ectopic expression of PTEN down-regulated EGR1 expression and restored dynamics of EGR1 expression in response to GM-CSF stimulation. Data from primary bone marrow cells from mice with Pten deletion further supports that PTEN is indispensible for myeloid leukemia cells in response to MAPK inhibitors. Finally, We demonstrate that the absence of EGR1 expression dynamics in response to GM-CSF stimulation is one of the mechanisms underlying drug resistance to MAPK inhibitors in leukemia cells with PTEN deficiency. Our data suggest a novel mechanism of PTEN in regulating expression of EGR1 in hematopoietic cells in response to cytokine stimulation. In conclusion, this study demonstrates that PTEN is dispensable for myeloid leukemia cells in response to MAPK inhibitors, and PTEN regulates EGR1 expression and contributes to the cytokine sensitivity in leukemia cells.     

Pharmacologic Blockade of JAK1/JAK2 Reduces GvHD and Preserves the Graft-Versus-Leukemia Effect

We have recently reported that interferon gamma receptor deficient (IFNγR−/−) allogeneic donor T cells result in significantly less graft-versus-host disease (GvHD) than wild-type (WT) T cells, while maintaining an anti-leukemia or graft-versus-leukemia (GvL) effect after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We demonstrated that IFNγR signaling regulates alloreactive T cell trafficking to GvHD target organs through expression of the chemokine receptor CXCR3 in alloreactive T cells. Since IFNγR signaling is mediated via JAK1/JAK2, we tested the effect of JAK1/JAK2 inhibition on GvHD. While we demonstrated that pharmacologic blockade of JAK1/JAK2 in WT T cells using the JAK1/JAK2 inhibitor, INCB018424 (Ruxolitinib), resulted in a similar effect to IFNγR−/− T cells both in vitro (reduction of CXCR3 expression in T cells) and in vivo (mitigation of GvHD after allo-HSCT), it remains to be determined if in vivo administration of INCB018424 will result in preservation of GvL while reducing GvHD. Here, we report that INCB018424 reduces GvHD and preserves the beneficial GvL effect in two different murine MHC-mismatched allo-HSCT models and using two different murine leukemia models (lymphoid leukemia and myeloid leukemia). In addition, prolonged administration of INCB018424 further improves survival after allo-HSCT and is superior to other JAK1/JAK2 inhibitors, such as TG101348 or AZD1480. These data suggest that pharmacologic inhibition of JAK1/JAK2 might be a promising therapeutic approach to achieve the beneficial anti-leukemia effect and overcome HLA-barriers in allo-HSCT. It might also be exploited in other diseases besides GvHD, such as organ transplant rejection, chronic inflammatory diseases and autoimmune diseases.