Thelma R. Tennant

Cytogenetic and genetic pathways in therapy-related acute myeloid leukemia

Therapy-related myelodysplastic syndrome and acute myeloid leukemia (t-MDS/t-AML) are late complications of cytotoxic therapy used in the treatment of malignant diseases. The most common subtype of t-AML ( approximately 75% of cases) develops after exposure to alkylating agents, and is characterized by loss or deletion of chromosome 5 and/or 7 [-5/del(5q), -7/del(7q)], and a poor outcome (median survival 8 months). In the University of Chicagos series of 386 patients with t-MDS/t-AML, 79 (20%) patients had abnormalities of chromosome 5, 95 (25%) patients had abnormalities of chromosome 7, and 85 (22%) patients had abnormalities of both chromosomes 5 and 7. t-MDS/t-AML with a -5/del(5q) is associated with a complex karyotype, characterized by trisomy 8, as well as loss of 12p, 13q, 16q22, 17p (TP53 locus), chromosome 18, and 20q. In addition, this subtype of t-AML is characterized by a unique expression profile (higher expression of genes) involved in cell cycle control (CCNA2, CCNE2, CDC2), checkpoints (BUB1), or growth (MYC), loss of expression of IRF8, and overexpression of FHL2. Haploinsufficiency of the RPS14, EGR1, APC, NPM1, and CTNNA1 genes on 5q has been implicated in the pathogenesis of MDS/AML. In previous studies, we determined that Egr1 acts by haploinsufficiency and cooperates with mutations induced by alkylating agents to induce myeloid leukemias in the mouse. To identify mutations that cooperate with Egr1 haploinsufficiency, we used retroviral insertional mutagenesis. To date, we have identified two common integration sites involving genes encoding transcription factors that play a critical role in hematopoiesis (Evi1 and Gfi1b loci). Of note is that the EVI1 transcription factor gene is deregulated in human AMLs, particularly those with -7, and abnormalities of 3q. Identifying the genetic pathways leading to t-AML will provide new insights into the underlying biology of this disease, and may facilitate the identification of new therapeutic targets.

Genome-wide association study to identify novel loci associated with therapy-related myeloid leukemia susceptibility

Therapy-related acute myeloid leukemia (t-AML) is a rare but fatal complication of cytotoxic therapy. Whereas sporadic cancer results from interactions between complex exposures and low-penetrance alleles, t-AML results from an acute exposure to a limited number of potent genotoxins. Consequently, we hypothesized that the effect sizes of variants associated with t-AML would be greater than in sporadic cancer, and, therefore, that these variants could be detected even in a modest-sized cohort. To test this, we undertook an association study in 80 cases and 150 controls using Affymetrix Mapping 10K arrays. Even at nominal significance thresholds, we found a significant excess of associations over chance; for example, although 6 associations were expected at P less than .001, we found 15 (P(enrich) = .002). To replicate our findings, we genotyped the 10 most significantly associated single nucleotide polymorphisms (SNPs) in an independent t-AML cohort (n = 70) and obtained evidence of association with t-AML for 3 SNPs in the subset of patients with loss of chromosomes 5 or 7 or both, acquired abnormalities associated with prior exposure to alkylator chemotherapy. Thus, we conclude that the effect of genetic factors contributing to cancer risk is potentiated and more readily discernable in t-AML compared with sporadic cancer.

Gain of MYC underlies recurrent trisomy of the MYC chromosome in acute promyelocytic leukemia

The leukemogenic effects of Myc drive recurrent trisomy in a mouse model of acute myeloid leukemia.

PML-RARα co-operates with Sox4 in acute myeloid leukemia development in mice

Acute promyelocytic leukemia is characterized by a chromosomal translocation involving the retinoic acid receptor alpha gene. To identify co-operating pathways to leukemogenesis, we crossed MRP8-PML/RARA transgenic mice with BXH-2 mice which harbor an endogenous murine leukemia virus that causes acute myeloid leukemia. Approximately half of the leukemias that arose in this cross showed features of acute promyelocytic leukemia. We identified 22 proviral insertion sites in acute promyelocytic-like leukemias and focused our analysis on insertion at Sox4, a HMG box transcription factor. Using a transplant model, co-operation between PML-RARα and Sox4 was confirmed with increased penetrance and reduced latency of disease. Interestingly, karyotypic analysis revealed cytogenetic changes suggesting that the factors combined to initiate but not complete leukemic transformation. The cooperation between these transcription factors is consistent with the paradigm of multiple routes to the disease and reinforces the concept that transcription factor networks are important therapeutic targets in myeloid leukemias.

The Clathrin-Binding Domain of CALM-AF10 Alters the Phenotype of Myeloid Neoplasms in Mice

The PICALM (CALM) gene, whose product is involved in clathrin-mediated endocytosis, has been identified in two recurring chromosomal translocations, involving either MLL or MLLT10 (AF10). We developed a mouse model of CALM-AF10+ leukemia to examine the hypothesis that disruption of endocytosis contributes to leukemogenesis. Exclusion of the C-terminal portion of CALM from the fusion protein, which is required for optimal binding to clathrin, resulted in the development of a myeloproliferative disease, whereas inclusion of this domain led to the development of acute myeloid leukemia and changes in gene expression of several cancer-related genes, notably Pim1 and Crebbp. Nonetheless, the development of leukemia could not be attributed directly to interference with endocytosis or consequential changes in proliferation and signaling. In leukemia cells, full-length CALM-AF10 localized to the nucleus with no consistent effect on growth factor endocyctosis, and suppressed histone H3 lysine 79 methylation regardless of the presence of clathrin. Using fluorescence resonance energy transfer analysis, we show that CALM-AF10 has a propensity to homo-oligomerize, raising the possibility that the function of endocytic proteins involved in chimeric fusions may be to provide dimerization properties, a recognized mechanism for unleashing oncogenic properties of chimeric transcription factors, rather than disrupting the internalization of growth factor receptors.

Inhibition of prostate cancer metastatic colonization by ∼4.2 Mb of human chromosome 12

Our previous studies demonstrate that introduction of a ∼70 cM region (now estimated at 63.75 Mb by the Human Genome Project) of human chromosome 12 into the highly metastatic Dunning rat prostate cancer cell line AT6.1 results in >30‐fold (≥90%) reduction in the number of overt metastases in spontaneous metastasis assays. We report the further localization and biological characterization of the metastasis‐suppressor activity encoded by a reduced region of chromosome 12. To localize this metastasis‐suppressor activity, a panel of AT6.1 microcell hybrids that retain varying portions of human chromosome 12 was constructed and subjected to sequence‐tagged site (STS)‐based PCR analysis and assessment of in vivo metastatic ability. Data from these complementary approaches localized the metastasis‐suppressor activity to a ∼4.2 Mb portion of human chromosome 12q24.3 comprised of 3 separate regions. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) and immunoblotting were used for differential expression analyses to identify which characterized genes, predicted gene sequences and expressed sequence tags (ESTs) within this region could be responsible for the observed metastasis suppression. Comprehensive in vivo studies showed that suppressed AT6.1‐12 hybrids that retain the metastasis‐suppressor region on 12q24.3 are capable of arriving at the secondary site, but are not able to persist there. Thus, unlike other metastasis‐suppressor genes characterized to date, the metastasis‐suppressor gene encoded by this region appears to utilize a different biologic mechanism to suppress the growth of overt metastases at the secondary site.

Northern and southern blotting and the polymerase chain reaction to detect gene expression.

For cancer cells to form a metastasis, cells from the primary tumor must overcome the local adhesive forces, migrate and invade the microcirculation, arrest at a secondary site, and then finally proliferate (1). As implied by its mult]step nature, cancer metastasis is a complex and dynamic process that is likely to be regulated by a series of genes at each step (2). A variety of approaches have been used to discern the molecular events that regulate this process. It is likely that the ability of a cancer cell to form clinically detectable metastases is influenced by a variety of factors, including alt]rations in the pattern of gene expression within the cancer cell. Such changes could be the result]of genetic or epigenetic modifications (3). Alt]ough there has been a growing emphasis on array-based techniques for high-throughput screening of gene expression patterns, there are several well established protocols that can be used to identify such molecular changes. This chapter describes two of these techniques: Northern and Southern blotting.E. M. Southern first described a method for immobilizing size-fractionated DNA fragments on a nitrocellulose membrane in 1975. Since then, a number of different variations of this blotting method have been developed, as well as a variety of ways by which scientists can generate and hybridize probes to detect specifically the sequences thus immobilized. Southern blotting is now a general term for a number of different methods by which DNA is transferred from a gel to a membrane, and because nitrocellulose is relatively fragile, improved membranes have been developed that are more durable and that have been optimized for allowing binding of nucleic acids.