Mahesh Shrestha

Inhibition of Rac GTPase signaling and downstream prosurvival Bcl-2 proteins as combination targeted therapy in MLL-AF9 leukemia.

The Rac family of small Rho GTPases coordinates diverse cellular functions in hematopoietic cells including adhesion, migration, cytoskeleton rearrangements, gene transcription, proliferation, and survival. The integrity of Rac signaling has also been found to critically regulate cellular functions in the initiation and maintenance of hematopoietic malignancies. Using an in vivo gene targeting approach, we demonstrate that Rac2, but not Rac1, is critical to the initiation of acute myeloid leukemia in a retroviral expression model of MLL-AF9 leukemogenesis. However, loss of either Rac1 or Rac2 is sufficient to impair survival and growth of the transformed MLL-AF9 leukemia. Rac2 is known to positively regulate expression of Bcl-2 family proteins toward a prosurvival balance. We demonstrate that disruption of downstream survival signaling through antiapoptotic Bcl-2 proteins is implicated in mediating the effects of Rac2 deficiency in MLL-AF9 leukemia. Indeed, overexpression of Bcl-xL is able to rescue the effects of Rac2 deficiency and MLL-AF9 cells are exquisitely sensitive to direct inhibition of Bcl-2 family proteins by the BH3-mimetic, ABT-737. Furthermore, concurrent exposure to NSC23766, a small-molecule inhibitor of Rac activation, increases the apoptotic effect of ABT-737, indicating the Rac/Bcl-2 survival pathway may be targeted synergistically.

AML cells are differentially sensitive to chemotherapy treatment in a human xenograft model

As acute myeloid leukemia (AML) xenograft models improve, the potential for using them to evaluate novel therapeutic strategies becomes more appealing. Currently, there is little information on using standard chemotherapy regimens in AML xenografts. Here we have characterized the immunodeficient mouse response to combined Ara-C (cytarabine) and doxorubicin treatment. We observed significant toxicity associated with doxorubicin that required optimization of the route of injection as well as the maximum-tolerated dose for immunodeficient strains. Mice treated with an optimized 5-day induction protocol showed transient weight loss, short-term reduction of peripheral blood cell and platelet counts, and slight anemia. Considerable cytotoxicity was observed in the bone marrow (BM), with primitive LSK cells having a significant survival advantage relative to more mature cells, consistent with the idea of chemotherapy targeting actively growing cells. Treated leukemic mice demonstrated reduced disease burden and increased survival, demonstrating efficacy. AML cells showed significantly increased sensitivity to doxorubicin-containing therapy compared with murine BM cells. Although early treatment could result in some cures, mice with significant leukemia grafts were not cured by using induction therapy alone. Overall, the data show that this model system is useful for the evaluation of novel chemotherapies in combination with standard induction therapy.

The thrombopoietin/MPL/Bcl-xL pathway is essential for survival and self-renewal in human preleukemia induced by AML1-ETO

AML1-ETO (AE) is a fusion product of translocation (8;21) that accounts for 40% of M2 type acute myeloid leukemia (AML). In addition to its role in promoting preleukemic hematopoietic cell self-renewal, AE represses DNA repair genes, which leads to DNA damage and increased mutation frequency. Although this latter function may promote leukemogenesis, concurrent p53 activation also leads to an increased baseline apoptotic rate. It is unclear how AE expression is able to counterbalance this intrinsic apoptotic conditioning by p53 to promote survival and self-renewal. In this report, we show that Bcl-xL is up-regulated in AE cells and plays an essential role in their survival and self-renewal. Further investigation revealed that Bcl-xL expression is regulated by thrombopoietin (THPO)/MPL-signaling induced by AE expression. THPO/MPL-signaling also controls cell cycle reentry and mediates AE-induced self-renewal. Analysis of primary AML patient samples revealed a correlation between MPL and Bcl-xL expression specifically in t(8;21) blasts. Taken together, we propose that survival signaling through Bcl-xL is a critical and intrinsic component of a broader self-renewal signaling pathway downstream of AML1-ETO-induced MPL.

UBASH3B/Sts-1-CBL axis regulates myeloid proliferation in human preleukemia induced by AML1-ETO

The t(8;21) rearrangement, which creates the AML1-ETO fusion protein, represents the most common chromosomal translocation in acute myeloid leukemia (AML). Clinical data suggest that CBL mutations are a frequent event in t(8;21) AML, but the role of CBL in AML1-ETO-induced leukemia has not been investigated. In this study, we demonstrate that CBL mutations collaborate with AML1-ETO to expand human CD34+ cells both in vitro and in a xenograft model. CBL depletion by shRNA also promotes the growth of AML1-ETO cells, demonstrating the inhibitory function of endogenous CBL in t(8;21) AML. Mechanistically, loss of CBL function confers hyper-responsiveness to thrombopoietin and enhances STAT5/AKT/ERK/Src signaling in AML1-ETO cells. Interestingly, we found the protein tyrosine phosphatase UBASH3B/Sts-1, which is known to inhibit CBL function, is upregulated by AML1-ETO through transcriptional and miR-9-mediated regulation. UBASH3B/Sts-1 depletion induces an aberrant pattern of CBL phosphorylation and impairs proliferation in AML1-ETO cells. The growth inhibition caused by UBASH3B/Sts-1 depletion can be rescued by ectopic expression of CBL mutants, suggesting that UBASH3B/Sts-1 supports the growth of AML1-ETO cells partly through modulation of CBL function. Our study reveals a role of CBL in restricting myeloid proliferation of human AML1-ETO-induced leukemia, and identifies UBASH3B/Sts-1 as a potential target for pharmaceutical intervention.

A FOXO1-induced oncogenic network defines the AML1-ETO preleukemic program

Understanding and blocking the self-renewal pathway of preleukemia stem cells could prevent acute myeloid leukemia (AML) relapse. In this study, we show that increased FOXO1 represents a critical mechanism driving aberrant self-renewal in preleukemic cells expressing the t(8;21)-associated oncogene AML1-ETO (AE). Although generally considered as a tumor suppressor, FOXO1 is consistently upregulated in t(8;21) AML. Expression of FOXO1 in human CD34+ cells promotes a preleukemic state with enhanced self-renewal and dysregulated differentiation. The DNA binding domain of FOXO1 is essential for these functions. FOXO1 activates a stem cell molecular signature that is also present in AE preleukemia cells and preserved in t(8;21) patient samples. Genome-wide binding studies show that AE and FOXO1 share the majority of their binding sites, whereby FOXO1 binds to multiple crucial self-renewal genes and is required for their activation. In agreement with this observation, genetic and pharmacological ablation of FOXO1 inhibited the long-term proliferation and clonogenicity of AE cells and t(8;21) AML cell lines. Targeting of FOXO1 therefore provides a potential therapeutic strategy for elimination of stem cells at both preleukemic and leukemic stages.

The full transforming capacity of MLL-Af4 is interlinked with lymphoid lineage commitment

Chromosome rearrangements involving the mixed-lineage leukemia gene (MLL) create MLL-fusion proteins, which could drive both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The lineage decision of MLL-fusion leukemia is influenced by the fusion partner and microenvironment. To investigate the interplay of fusion proteins and microenvironment in lineage choice, we transplanted human hematopoietic stem and progenitor cells (HSPCs) expressing MLL-AF9 or MLL-Af4 into immunodeficient NSGS mice, which strongly promote myeloid development. Cells expressing MLL-AF9 efficiently developed AML in NSGS mice. In contrast, MLL-Af4 cells, which were fully oncogenic under lymphoid conditions present in NSG mice, displayed compromised transformation capacity in a myeloid microenvironment. MLL-Af4 activated a self-renewal program in a lineage-dependent manner, showing the leukemogenic activity of MLL-Af4 was interlinked with lymphoid lineage commitment. The C-terminal homology domain (CHD) of Af4 was sufficient to confer this linkage. Although the MLL-CHD fusion protein failed to immortalize HSPCs in myeloid conditions in vitro, it could successfully induce ALL in NSG mice. Our data suggest that defective self-renewal ability and leukemogenesis of MLL-Af4 myeloid cells could contribute to the strong B-cell ALL association of MLL-AF4 leukemia observed in the clinic.

Supraphysiologic levels of the AML1-ETO isoform AE9a are essential for transformation.

Significance The AE9a protein (alternative splicing at exon 9) is often used to model t(8;21) leukemia. Our study demonstrates that increased oncogene dosage is a critical parameter of AE9a transformation, likely as a result of impaired transcriptional regulation of AML1-ETO target genes. This insight could assist in identifying those downstream genes most critical for t(8;21)-associated transformation. Chromosomal translocation 8;21 is found in 40% of the FAB M2 subtype of acute myeloid leukemia (AML). The resultant in-frame fusion protein AML1-ETO (AE) acts as an initiating oncogene for leukemia development. AE immortalizes human CD34+ cord blood cells in long-term culture. We assessed the transforming properties of the alternatively spliced AE isoform AE9a (or alternative splicing at exon 9), which is fully transforming in a murine retroviral model, in human cord blood cells. Full activity was realized only upon increased fusion protein expression. This effect was recapitulated in the AE9a murine AML model. Cotransduction of AE and AE9a resulted in a strong selective pressure for AE-expressing cells. In the context of AE, AE9a did not show selection for increased expression, affirming observations of human t(8;21) patient samples where full-length AE is the dominant protein detected. Mechanistically, AE9a showed defective transcriptional regulation of AE target genes that was partially corrected at high expression. Together, these results bring an additional perspective to our understanding of AE function and highlight the contribution of oncogene expression level in t(8;21) experimental models.

MAPK8-mediated stabilization of SP1 is essential for RUNX1-RUNX1T1 – driven leukaemia

The presence of the RUNX1-RUNX1T1 (AML1-ETO) fusion protein indicates acute myeloid leukaemia (AML) with t(8;21)(q22;q22), which accounts for approximately 4% of all cases of AML and 40% of all cases of French-American-British M2 type AML. This product is the consequence of a reciprocal translocation that fuses the DNA-binding domain of the RUNX1 transcription factor – essential for transcriptional activation of genes involved in normal haematopoiesis – in frame with nearly the entire RUNX1T1 transcriptional repressor protein. In human haematopoietic stem and progenitor cells (HSPC), RUNX1-RUNX1T1 promotes selfrenewal and interferes with efficient myeloid and erythroid differentiation, although it is not sufficient to cause leukaemic transformation (Mulloy et al, 2002). Several studies have reinforced the importance of the interaction between RUNX1-RUNX1T1 and other transcription factors on its DNA-binding profile, and we previously identified the Sp1 transcription factor (SP1) binding site in more than 50% of DNA-bound RUNX1-RUNX1T1 protein (Maiques-Diaz et al, 2012). SP1 induces the expression of essential haematopoietic genes (i.e. KIT, DNMT1) and forms part of the SP1/NF-jB/HDAC1/MIR29B network (Liu et al, 2008, 2010). It also controls CDKN1A expression depending on and independently of TP53 binding (Huang et al, 2000; Koutsodontis et al, 2001). In the present study, we further explored the importance of the SP1 protein in the RUNX1RUNX1T1 leukaemogenic process and found that not only is SP1 directly regulated by the fusion protein but that it is also a key factor for RUNX1-RUNX1T1 AML cell maintenance. Details of the materials and methods are provided in Data S1. We previously reported relatively low SP1 mRNA levels in human haematopoetic stem progenitor cells expressing RUNX1-RUNX1T1 (referred to hereafter as HSPC-RR) and the SKNO1 cell line compared with HSPC (selected CD34 cells with an empty vector transduction) (Maiques-Diaz et al, 2012). However, abundant SP1 protein levels were observed in RUNX1-RUNX1T1 – expressing cells compared with normal HSPC (Fig 1A). To determine whether SP1 protein accumulation was a direct effect of RUNX1-RUNX1T1 expression, we selected normal CD34 cells and transduced them with RUNX1-RUNX1T1 – expressing retroviruses (for construct information, see Mulloy et al, 2002). A significant increase in SP1 protein levels was observed 1 week after transduction (Fig 1B), confirming that SP1 protein accumulates as a direct effect of RUNX1-RUNX1T1 expression in HSPCs. SP1 protein regulation is mediated by a variety of posttranslational modifications that have been reported to control SP1 protein levels, transactivation and DNA binding affinity. MAPK8 (also termed JNK1) is known to impair SP1 proteasomal degradation by inducing phosphorylation of Thr278 and Thr739 (Chuang et al, 2008). Interestingly, high levels of MAPK8 mRNA and MAPK8 protein relative to HSPC cells were observed in HSPC-RR and SKNO1 (Fig 1A and C). We analysed several AML cell lines harbouring different chromosomal translocations and, with the exception of the BCR-ABL1–positive K562 cell line, none showed high SP1 protein levels (Fig S1A), suggesting that this event is associated with specific AML contexts. Increased MAPK8 activation (measured as phospho-MAPK8) was also observed in both SKNO1 and K562 cells, correlating with the higher SP1 levels observed (Fig S1A). To further investigate the role of MAPK8 in SP1 stabilization in this cellular context, we treated SKNO1 cells with JNK/MAPK8 inhibitor II (SP600125; Calbiochem, San Diego, CA, USA), which competes reversibly with ATP, abrogating MAPK8 and MAPK9 (also termed JNK2) activity. Two days’ exposure of SKNO1 cells with 12 or 25 lmol/l of MAPK inhibitor (MAPKi) led to a significant reduction in levels of SP1, especially of the non-phosphorylated form, not impaired for proteasomal degradation (Fig 1D). Washing out the compound returned SP1 levels to normal (Fig 1D). Interestingly, RUNX1-RUNX1T1 – expressing cells were significantly more sensitive to JNK inhibition than the THP1 AML cell line, which is driven by the expression of the KMT2AMLLT3 (MLL-AF9) fusion gene and showed very low SP1 levels (Fig S2B). After 24 h of treatment with 25 lmol/l of MAPKi, less than 40% of RUNX1-RUNX1T1 cells were viable, whereas 80% of THP1 cells remained alive (Fig 1E). The biological importance of SP1 in t(8;21) leukaemia was further studied using two lentiviral short hairpin constructs on three independent HSPC-RR clones. After antibiotic selection, the cells showed a complete reduction in viable SP1 knockdown cells, which was not observed in the non-targeting transduced HSPC-RR clones (Fig S2A). Moreover, the stable knockdown of SP1 in the SKNO1 cell line (Fig 2A) resulted in significantly reduced growth and increased apoptosis (Fig 2B–C and S2B). When the same SP1 shRNA constructs were used in THP1 cells, SP1 knockdown had no effect in this cellular context (Fig S2C–D), indicating that the effect is directly related to RUNX1-RUNX1T1 expression. correspondence