Linda Wolff

Response and resistance to MEK inhibition in leukaemias initiated by hyperactive Ras

The cascade comprising Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) is a therapeutic target in human cancers with deregulated Ras signalling, which includes tumours that have inactivated the Nf1 tumour suppressor. Nf1 encodes neurofibromin, a GTPase-activating protein that terminates Ras signalling by stimulating hydrolysis of Ras–GTP. We compared the effects of inhibitors of MEK in a myeloproliferative disorder (MPD) initiated by inactivating Nf1 in mouse bone marrow and in acute myeloid leukaemias (AMLs) in which cooperating mutations were induced by retroviral insertional mutagenesis. Here we show that MEK inhibitors are ineffective in MPD, but induce objective regression of many Nf1-deficient AMLs. Drug resistance developed because of outgrowth of AML clones that were present before treatment. We cloned clone-specific retroviral integrations to identify candidate resistance genes including Rasgrp1, Rasgrp4 and Mapk14, which encodes p38α. Functional analysis implicated increased RasGRP1 levels and reduced p38 kinase activity in resistance to MEK inhibitors. This approach represents a robust strategy for identifying genes and pathways that modulate how primary cancer cells respond to targeted therapeutics and for probing mechanisms of de novo and acquired resistance.

Hematopoiesis and leukemogenesis in mice expressing oncogenic NrasG12D from the endogenous locus

NRAS is frequently mutated in hematologic malignancies. We generated Mx1-Cre, Lox-STOP-Lox (LSL)-Nras(G12D) mice to comprehensively analyze the phenotypic, cellular, and biochemical consequences of endogenous oncogenic Nras expression in hematopoietic cells. Here we show that Mx1-Cre, LSL-Nras(G12D) mice develop an indolent myeloproliferative disorder but ultimately die of a diverse spectrum of hematologic cancers. Expressing mutant Nras in hematopoietic tissues alters the distribution of hematopoietic stem and progenitor cell populations, and Nras mutant progenitors show distinct responses to cytokine growth factors. Injecting Mx1-Cre, LSL-Nras(G12D) mice with the MOL4070LTR retrovirus causes acute myeloid leukemia that faithfully recapitulates many aspects of human NRAS-associated leukemias, including cooperation with deregulated Evi1 expression. The disease phenotype in Mx1-Cre, LSL-Nras(G12D) mice is attenuated compared with Mx1-Cre, LSL-Kras(G12D) mice, which die of aggressive myeloproliferative disorder by 4 months of age. We found that endogenous Kras(G12D) expression results in markedly elevated Ras protein expression and Ras-GTP levels in Mac1(+) cells, whereas Mx1-Cre, LSL-Nras(G12D) mice show much lower Ras protein and Ras-GTP levels. Together, these studies establish a robust and tractable system for interrogating the differential properties of oncogenic Ras proteins in primary cells, for identifying candidate cooperating genes, and for testing novel therapeutic strategies.

Oncogenic activation of c-Myb by carboxyl-terminal truncation leads to decreased proteolysis by the ubiquitin-26S proteasome pathway

c-myb activation by insertional mutagenesis in murine myeloid leukemias can lead to amino (NH2)-terminal or carboxyl (COOH)-terminal truncation of its protein product. We observed that in these leukemias, the steady state level of the protein truncated at the COOH terminus was remarkably higher than that of the protein truncated at the NH2-terminus or full length wild-type protein. To examine the rate of proteolysis of different forms of Myb in a uniform cellular background, the proteins were constitutively expressed in the myeloblast cell line M1, using the retrovirus vector LXSN. In pulse chase experiments, using metabolically 35S-labeled proteins, it was determined that COOH-terminal truncation of c-Myb by 248 aa (CT-c-Myb) substantially increases protein stability, resulting in a t1/2 of about 140 min, as compared to 50 min for full length c-Myb (FL-c-Myb). In an investigation of the mechanism involved in the in vivo degradation of this short lived transcription factor, inhibitors of the lysosomal (chloroquine), proteasomal (ALLM, ALLN, lactacystin) and calpains (EGTA, E-64d, BAPTA/AM) pathways were utilized. Results of this experiment identified the 26S proteasome as a major pathway responsible for rapid breakdown of the protein in hematopoietic cells. Further experiments carried out in vitro demonstrated that c-Myb can be ubiquitinated, suggesting that this process may be involved in the targeting of wild-type c-Myb to degradation by the 26S proteasome. In addition, it was demonstrated that CT-c-Myb was less efficiently ubiquitinated than wild-type protein indicating that defects in modification account for its escape from rapid turnover. We speculate that the increased half-life of c-Myb resulting from truncation could contribute to its transforming potential.

Retroviral Insertional Mutagenesis Identifies Genes that Collaborate with NUP98-HOXD13 during Leukemic Transformation

The t(2;11)(q31;p15) chromosomal translocation results in a fusion between the NUP98 and HOXD13 genes and has been observed in patients with myelodysplastic syndrome (MDS) or acute myelogenous leukemia. We previously showed that expression of the NUP98-HOXD13 (NHD13) fusion gene in transgenic mice results in an invariably fatal MDS; approximately one third of mice die due to complications of severe pancytopenia, and about two thirds progress to a fatal acute leukemia. In the present study, we used retroviral insertional mutagenesis to identify genes that might collaborate with NHD13 as the MDS transformed to an acute leukemia. Newborn NHD13 transgenic mice and littermate controls were infected with the MOL4070LTR retrovirus. The onset of leukemia was accelerated, suggesting a synergistic effect between the NHD13 transgene and the genes neighboring retroviral insertion events. We identified numerous common insertion sites located near protein-coding genes and confirmed dysregulation of a subset of these by expression analyses. Among these genes were Meis1, a known collaborator of HOX and NUP98-HOX fusion genes, and Mn1, a transcriptional coactivator involved in human leukemia through fusion with the TEL gene. Other putative collaborators included Gata2, Erg, and Epor. Of note, we identified a common insertion site that was >100 kb from the nearest coding gene, but within 20 kb of the miR29a/miR29b1 microRNA locus. Both of these miRNA were up-regulated, demonstrating that retroviral insertional mutagenesis can target miRNA loci as well as protein-coding loci. Our data provide new insights into NHD13-mediated leukemogenesis as well as retroviral insertional mutagenesis mechanisms.

Mutant Ikzf1, KrasG12D, and Notch1 cooperate in T lineage leukemogenesis and modulate responses to targeted agents.

Mice that accurately model the genetic diversity found in human cancer are valuable tools for interrogating disease mechanisms and investigating novel therapeutic strategies. We performed insertional mutagenesis with the MOL4070LTR retrovirus in Mx1-Cre, KrasG12D mice and generated a large cohort of T lineage acute lymphoblastic leukemias (T-ALLs). Molecular analysis infers that retroviral integration within Ikzf1 is an early event in leukemogenesis that precedes KrasG12D expression and later acquisition of somatic Notch1 mutations. Importantly, biochemical analysis uncovered unexpected heterogeneity, which suggests that Ras signaling networks are remodeled during multistep tumorigenesis. We tested tumor-derived cell lines to identify biomarkers of therapeutic response to targeted inhibitors. Whereas all T-ALLs tested were sensitive to a dual-specificity phosphoinosityl 3-kinase/mammalian target of rapamycin inhibitor, biochemical evidence of Notch1 activation correlated with sensitivity to γ-secretase inhibition. In addition, KrasG12D T-ALLs were more responsive to a MAP/ERK kinase inhibitor in vitro and in vivo. Together, these studies identify a genetic pathway involving Ikzf1, KrasG12D, and Notch1 in T lineage leukemogenesis, reveal unexpected diversity in Ras-regulated signaling networks, and define biomarkers of drug responses that may inform treatment strategies.

A Moloney murine leukemia virus-based retrovirus with 4070A long terminal repeat sequences induces a high incidence of myeloid as well as lymphoid neoplasms.

ABSTRACT Retroviruses can be used to accelerate hematopoietic cancers predisposed to neoplastic disease by prior genetic manipulations such as in transgenic or knockout mice. The virus imparts a second neoplastic “hit,” providing evidence that the initial hit is transforming. In the present study, a unique retrovirus was developed that can induce a high incidence of myeloid disease and has a broad host range. This agent is a Moloney murine leukemia virus (Mo-MuLV)-based virus that has most of the U3 region of the long terminal repeat (LTR) replaced with that of retrovirus 4070A. Like Mo-MuLV, this virus, called MOL4070LTR, is NB-tropic and not restricted by Fv1 allelles. MOL4070LTR causes myeloid leukemias in ca. 50% of mice, a finding in contrast to Mo-MuLV, which induces almost exclusively lymphoid disease. The data suggest that the LTR of the 4070A virus expands the tissue tropism of the disease to the myeloid lineage. Interesting, MCF recombinant envelope was expressed in the lymphoid but not the myeloid neoplasms of BALB/c mice. This retrovirus has the potential for accelerating myeloid disease in genetically engineered mice.

Haploinsufficiency of SAMD9L, an endosome fusion facilitator, causes myeloid malignancies in mice mimicking human diseases with monosomy 7.

Monosomy 7 and interstitial deletion of 7q (-7/7q-) are well-recognized nonrandom chromosomal abnormalities frequently found among patients with myelodysplastic syndromes (MDSs) and myeloid leukemias. We previously identified candidate myeloid tumor suppressor genes (SAMD9, SAMD9-like = SAMD9L, and Miki) in the 7q21.3 subband. We established SAMD9L-deficient mice and found that SAMD9L(+/-) mice as well as SAMD9L(-/-) mice develop myeloid diseases resembling human diseases associated with -7/7q-. SAMD9L-deficient hematopoietic stem cells showed enhanced colony formation potential and in vivo reconstitution ability. SAMD9L localizes in early endosomes. SAMD9L-deficient cells showed delays in homotypic endosome fusion, resulting in persistence of ligand-bound cytokine receptors. These findings suggest that haploinsufficiency of SAMD9L and/or SAMD9 gene(s) contributes to myeloid transformation.

Stress-induced Inactivation of the c-Myb Transcription Factor through Conjugation of SUMO-2/3 Proteins

Post-translational modifications, such as phosphorylation, acetylation, ubiquitination, and SUMOylation, play an important role in regulation of the stability and the transcriptional activity of c-Myb. Conjugation of small ubiquitin-like modifier type 1 (SUMO-1) to lysines in the negative regulatory domain strongly suppresses its transcriptional activity. Here we report conjugation of two other members of the SUMO protein family, SUMO-2 and SUMO-3, and provide evidence that this post-translational modification negatively affects transcriptional activity of c-Myb. Conjugation of SUMO-2/3 proteins is strongly enhanced by several different cellular stresses and occurs primarily on two lysines, Lys523 and Lys499. These lysines are in the negative regulatory domain of c-Myb and also serve as acceptor sites for SUMO-1. Stress-induced SUMO-2/3 conjugation is very rapid and independent of activation of stress-activated protein kinases of the SAPK and JNK families. PIAS-3 protein was identified as a new c-Myb-specific SUMO-E3 ligase that both catalyzes conjugation of SUMO-2/3 proteins to c-Myb and exerts a negative effect on c-Myb-induced reporter gene activation. Interestingly, co-expression of a SPRING finger mutant of PIAS-3 significantly suppresses SUMOylation of c-Myb under stress. These results argue that PIAS-3 SUMO-E3 ligase plays a critical role in stress-induced conjugation of SUMO-2/3 to c-Myb. We also detected stress-induced conjugation of SUMO-2/3 to c-Myb in hematopoietic cells at the levels of endogenously expressed proteins. Furthermore, according to the negative role of SUMO conjugation on c-Myb capacity, we have observed rapid stress-induced down-regulation of the targets genes c-myc and bcl-2 of c-Myb. Our findings demonstrate that SUMO-2/3 proteins conjugate to c-Myb and negatively regulate its activity in cells under stress.

Hypermethylation of the Ink4b locus in murine myeloid leukemia and increased susceptibility to leukemia in p15(Ink4b)-deficient mice.

The Ink4b gene (Cdkn2b) encodes p15Ink4b, a cyclin-dependent kinase inhibitor. It has been implicated in playing a role in the development of acute myeloid leukemia (AML) in man, since it is hypermethylated with high frequency. We provide evidence that the gene is a tumor suppressor for myeloid leukemia in mice. The evidence is twofold: (1) retrovirus-induced myeloid leukemias of the myelomonocytic phenotype were found to have hypermethylation of the 5′ CpG island of the Ink4b gene, and this could be correlated with reduced mRNA expression, as demonstrated by TaqMan real-time PCR. p15Ink4b mRNA expression in a leukemia cell line, with hypermethylation at the locus, was induced following treatment with 5-aza-2′-deoxycytidine. (2) Targeted deletion of one allele in mice by removal of exon 2 increases their susceptibility to retrovirus-induced myeloid leukemia. Mice deficient in both alleles were not more susceptible to myeloid disease than those deficient in one allele, raising the possibility that there are opposing forces related to the development of myeloid leukemia in Ink4b null mice.

Challenges in vector and trial design using retroviral vectors for long-term gene correction in hematopoietic stem cell gene therapy.

Over the past two decades, incredible progress has been made using gene therapy for inherited severe immunodeficiency disorders, such as X-linked severe combined immunodeficiency disorder (SCID-X1) and adenosine deaminase deficiency–severe combined immunodeficiency disorder (ADA-SCID).1,2,3 However, for reasons that remain unclear, gene transfer for SCID-X1 has also been associated with some cases of vector-induced leukemia whereas no cases have been seen in the ADA-SCID trials despite the common use of g-retroviral vectors. The first case was reported in a French gene transfer trial for SCID-X1.4 Over the next six years, an additional three cases were reported in that trial and one in a second SCID-X1 trial that enrolled a combined total of 20 subjects.2 Unfortunately, genotoxicity would not remain confined to SCID-X1. Recent reports of insertional mutagenesis leading to myelodysplastic syndrome in a trial for chronic granulomatous disease and a case of leukemia in a trial for Wiskott-Aldrich syndrome (WAS), both of which used g-retroviral vectors, underscored that this type of toxicity can also apply to other disease settings.5,6,7 In all these cases, insertion of the g-retroviral vector near known proto-oncogenes led to enhancer-mediated expression of these proto-oncogenes.