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Mechanistic target of rapamycin (MTOR) is a highly conserved serine/threonine kinase that critically regulates cell growth, proliferation, differentiation, and survival. Previously, we have implicated Mtor as a plasmacytoma-resistance locus, Pctr2 , in mice. Here, we report that administration of the tumor-inducing agent pristane decreases Mtor gene expression to a greater extent in mesenteric lymph nodes of BALB/cAnPt mice than of DBA/2N mice. We identified six allelic variants in the Mtor promoter region in BALB/cAnPt and DBA/2N mice. To determine the effects of these variants on Mtor transcription, we constructed a series of luciferase reporters containing these promoter variants and transfected them into mouse plasmacytoma cells. We could attribute the differences in Mtor promoter activity between the two mouse strains to a C → T change at the -6 position relative to the transcriptional start site Tssr 40273; a T at this position in the BALB promoter creates a consensus binding site for the transcription factor MZF1 (myeloid zinc finger 1). Results from electrophoretic mobility shift assays and DNA pulldown assays with ChIP-PCR confirmed that MZF1 binds to the cis -element TGGGGA located in the -6/-1 Mtor promoter region. Of note, MZF1 significantly and differentially down-regulated Mtor promoter activity, with MZF1 overexpression reducing Mtor expression more strongly in BALB mice than in DBA mice. Moreover, MZF1 overexpression reduced Mtor expression in both fibroblasts and mouse plasmacytoma cells, and Mzf1 knockdown increased Mtor expression in BALB3T3 and NIH3T3 fibroblast cells. Our results provide evidence that MZF1 down-regulates Mtor expression in pristane-induced plasmacytomas in mice.

Enhanced proliferative signaling and loss of cell cycle regulation are essential for cancer progression. Increased mitogenic signaling through activation of the mTOR pathway, coupled with deregulation of the Cyclin D/retinoblastoma (Rb) pathway is a common feature of lymphoid malignancies, including plasmacytoma (PCT), multiple myeloma (MM), Burkitt's lymphoma (BL), and mantle cell lymphoma (MCL). Here we evaluate the synergy of pharmacologically affecting both of these critical pathways using the mTOR inhibitor sirolimus and the histone deacetylase inhibitor entinostat. A dose-matrix screening approach found this combination to be highly active and synergistic in a panel of genetically diverse human MM cell lines. Synergy and activity was observed in mouse PCT and human BL and MCL cell lines tested in vitro, as well as in freshly isolated primary MM patient samples tested ex vivo. This combination had minimal effects on healthy donor cells and retained activity when tested in a co-culture system simulating the protective interaction of cancer cells with the tumor microenvironment. Combining sirolimus with entinostat enhanced cell cycle arrest and apoptosis. At the molecular level, entinostat increased the expression of cell cycle negative regulators including CDKN1A (p21) and CDKN2A (p16), while the combination decreased critical growth and survival effectors including Cyclin D, BCL-XL, BIRC5, and activated MAPK., (Published by Elsevier B.V.)

The human HIN-200 gene cluster and its mouse counterpart, the interferon inducible-200 (Ifi200) family, both on Chr 1, are associated with several diseases, including solid tumors and lupus. Our study was initiated to identify the modifier gene(s) encoded by the Pctm locus, in which mouse B-cell plasmacytomas induced by pristane are associated with heterozygosity of Chr 1 genes near the Ifi200 cluster. A screen for differentially expressed genes in granulomatous tissues induced by pristane in resistant and susceptible strains identified a new Ifi200 member whose expression was 1000-fold higher in the strain carrying the resistant allele of Pctm and was the most highly expressed Ifi200 gene. The gene, designated Mndal (for MNDA-like, myeloid nuclear differentiation antigen-like), was absent in the susceptible genome, as were genomic sequences upstream of Ifi203, the gene adjacent to Mndal. Ectopic expression of MNDAL suppressed cell growth, which, together with the disease susceptibility of heterozygotes at the Pctm locus, suggests that Mndal, perhaps with Ifi203, acts as a tumor suppressor and display(s) haploinsufficiency. Mndal is highly polymorphic among inbred mouse strains, because it is absent in 10 of 24 strains. This polymorphism may have implications for other disease modifiers mapping to the same region.

Susceptibility to mouse plasmacytomagenesis is a complex genetic trait controlled by several Pctr loci (Pctr1, Pctr2, etc). Congenic strain analysis narrowed the genetic interval surrounding the Pctr2 locus, and genes identified in the interval were sequenced from susceptible BALB/c and resistant DBA/2 mice. Frap (FKBP12 rapamycin-associated protein, mTOR, RAFT) was the only gene differing in amino acid sequence between alleles that correlated with strain sensitivity to tumor development. The in vitro kinase activity of the BALB/c FRAP allele was lower than the DBA/2 allele; phosphorylation of p53 and PHAS1/4EBP1 (properties of heat and acid stability/eukaryotic initiation factor 4E-binding protein) and autophosphorylation of FRAP were less efficient with the BALB/c allele. FRAP also suppressed transformation of NIH 3T3 cells by ras, with DBA/2 FRAP being more efficient than BALB/c FRAP. Rapamycin, a specific inhibitor of FRAP, did not inhibit growth of plasmacytoma cell lines. These studies identify Frap as a candidate tumor suppressor gene, in contrast to many reports that have focused on its prooncogenic properties. Frap may be similar to Tgfb and E2f in exerting both positive and negative growth-regulatory signals, depending on the timing, pathway, or tumor system involved. The failure of rapamycin to inhibit plasma cell tumor growth suggests that FRAP antagonists may not be appropriate for the treatment of plasma cell tumors. Pctr2 joins Pctr1 in possessing alleles that modify susceptibility to plasmacytomagenesis by encoding differences in efficiency of function (efficiency alleles), rather than all-or-none, gain-of-function, or loss-of-function alleles. By analogy, human cancer may also result from the combined effects of several inefficient alleles.

The susceptibility of BALB/c mice to pristane-induced plasmacytomas is a complex genetic trait involving multiple loci, while DBA/2 and C57BL/6 strains are genetically resistant to the plasmacytomagenic effects of pristane. In this model system for human B-cell neoplasia, one of the BALB/c susceptibility and modifier loci, Pctr1, was mapped to a 5.7-centimorgan (cM) chromosomal region that included Cdkn2a, which encodes p16(INK4a) and p19(ARF), and the coding sequences for the BALB/c p16(INK4a) and p19(ARF) alleles were found to be polymorphic with respect to their resistant Pctr1 counterparts in DBA/2 and C57BL/6 mice (45). In the present study, alleles of Pctr1, Cdkn2a, and D4Mit15 from a resistant strain (BALB/cDAG) carrying DBA/2 chromatin were introgressively backcrossed to the susceptible BALB/c strain. The resultant C.DAG-Pctr1 Cdkn2a D4Mit15 congenic was more resistant to plasmacytomagenesis than BALB/c, thus narrowing Pctr1 to a 1.5-cM interval. Concomitantly, resistant C57BL/6 mice, from which both gene products of the Cdkn2a gene have been eliminated, developed pristane-induced plasma cell tumors over a shorter latency period than the traditionally susceptible BALB/cAn strain. Biological assays of the p16(INK4a) and p19(ARF) alleles from BALB/c and DBA/2 indicated that the BALB/c p16(INK4a) allele was less active than its DBA/2 counterpart in inducing growth arrest of mouse plasmacytoma cell lines and preventing ras-induced transformation of NIH 3T3 cells, while the two p19(ARF) alleles displayed similar potencies in both assays. We propose that the BALB/c susceptibility/modifier locus, Pctr1, is an "efficiency" allele of the p16(INK4a) gene.

Plasma cell tumor induction in mice by pristane is under multigenic control. BALB/c mice are susceptible to tumor development; whereas DBA/2 mice are resistant. Restriction fragment length polymorphisms between BALB/c and DBA/2 for Cdkn2a(p16) and Cdkn2b(p15), and between BALB/c and Mus spretus for Cdkn2c(p18(INK4c)) were used to position these loci with respect to the Pctr1 locus. These cyclin-dependent kinase (CDK) inhibitors mapped to a 6 cM interval of chromosome 4 between Ifna and Tal1. C.D2-Chr 4 congenic strains harboring DBA/2 alleles associated with the Pctr1 locus contained DBA/2 "resistant" alleles of the CDK4/CDK6 inhibitors p16 and p15. On sequencing p16 and p18 cDNAs, two different allelic variants within ankyrin repeat regions of p16 were found between BALB/c and DBA/2 mice. By using an assay involving PCR amplification and restriction enzyme digestion, allelic variants were typed among several inbred strains of mice. One of the variants, G232A, was specific to two inbred strains, BALB/cAn and ABP/Le, of mice and occurred in a highly conserved amino acid in both human and rat p16. When tested with wild-type (DBA/2) p16, both A134C and G232A BALB/c-specific variants of p16 were inefficient in their ability to inhibit the activity of cyclin D2/CDK4 in kinase assays with retinoblastoma protein, suggesting this defective, inherited allele plays an important role in the genetic susceptibility of BALB/c mice for plasmacytoma induction and that p16(INK4a) is a strong candidate for the Pctr1 locus.

Mouse plasmacytomas share pathogenetic features in common with both multiple myeloma and Burkitt's lymphoma in humans. Susceptibility to plasmacytoma induction by intraperitoneal pristane in mice is controlled by multiple genes. At least two of these genes reside on mouse chromosome 4 in regions of the genome sharing linkage homology with human chromosomes 9p21, 1p32, and 1p36. A series of congenic strains recombinant for regions of mouse chromosome 4 in the vicinity of the Pctr2 predisposition locus were created and typed for their tumor susceptibility/resistance phenotypes. These strains were derived by introgressively backcrossing alleles from resistant DBA/2 mice onto the susceptible BALB/cAnPt background. Six resistant and two susceptible strains were allelotyped for 10 genes and 49 random DNA markers to identify the smallest region of overlap in the resistant strains. These studies have determined that the Pctr2 locus resides in either a 500-kb interval proximal to Nppa, or in a 1- to 2-centiMorgan (cM) interval distal to Nppa. In these congenic strain analyses, the Nppa and Fv1 loci, in addition to genes within about 1 cM of these loci, have been excluded as candidates for the Pctr2 locus. A relevant locus that may reside in this interval is Rep2; it is associated with the efficiency of repairing X-ray induced DNA damage sustained during the G2 phase of the mitotic cycle. The Pctr2 locus acts in a codominant fashion. F1 hybrids between resistant and susceptible congenic strains exhibit a reduced tumor incidence and a significant delay in the onset of tumorigenesis. Identification and eventual cloning of the Pctr2 locus may assist in the identification of genes involved in many types of cancer showing aberrations in human chromosome 1p36.

Interleukin (IL) 6 has been suggested to be the major cytokine responsible for proliferation of neoplastic plasma cells in both human myeloma and mouse plasmacytoma. Much of the evidence supporting this suggestion is derived from in vitro studies in which the survival or proliferation of some plasma cell tumors has been found to be IL-6 dependent. However, it remains unclear whether this dependency is the consequence of in vivo or in vitro selective pressures that preferentially expand IL-6-responsive tumor cells, or whether it reflects a critical in vivo role for IL-6 in plasma cell neoplasia. To address this question, we have attempted to induce plasma cell tumors in normal mice and in IL-6-deficient mice generated by introduction of a germline-encoded null mutation in the IL-6 gene. The results demonstrate that mice homozygous (+/+) or heterozygous (+/-) for the wild-type IL-6 allele yield the expected incidences of plasma cell tumors. In contrast, mice homozygous for the IL-6-null allele (-/-) are completely resistant to plasma cell tumor development. These studies define the essential role of IL-6 in the development of B lineage tumors in vivo and provide experimental support for continued efforts to modulate this cytokine in the treatment of appropriate human B cell malignancies.

Trisomy of chromosome 11 (Ts11) is the second most frequent nonrandom chromosomal change in murine plasmacytomas (PCTs). The frequency of Ts11 is significantly higher in PCTs induced in pristane-conditioned mice infected by Abelson-murine leukemia virus (52%) compared to those induced by pristane alone (8.1%). Although the significance of Ts11 in mouse plasmacytomagenesis is not clearly understood it is hypothesized that a gene or genes located on chromosome (Chr) 11 may specifically promote the development of PCTs in which both oncogenes, c-myc and v-abl, are abundantly expressed. To test this assumption we induced PCTs by three highly effective plasmacytomagenic retroviruses: ABL-MYC, J3V1, and RIM. Nearly 90% of PCTs that arose in BALB/c, (BALB/c x DBA/2N)F1, BALB/c-nu/nu, and 5-month-old SCID mice infected with ABL-MYC virus were trisomic for Chr 11. In contrast, < 10% of PCTs induced by J3V1 or RIM retroviral constructs encompassing either v-myc and v-raf or c-myc and v-Ha-ras oncogenes, respectively, contained Ts11. We have also investigated whether the entire Chr 11 or any particular subregion is preferentially duplicated in the process of ABL-MYC plasmacytomagenesis. By inducing PCTs in F1 heterozygous mice that are carriers of reciprocal translocations involving Chr 11 we found that the duplicated chromosomal region is located distal to the T4Dn breakpoint (11B5 band) on the telomeric segment of Chr 11. The regular duplication of this chromosomal segment strongly suggests the presence of a gene or genes whose amplification is of critical importance for v-abl associated murine plasmacytomagenesis.

BALB/cAn mice are highly susceptible to the induction of plasmacytomas (PCTs) by the i.p. injection of paraffin oils, whereas DBA/2 mice are solidly resistant. To search for genes that control the dominant resistant phenotype of DBA/2, BALB/c.DBA/2 (C.D2) congenic strains were constructed, and the susceptibility and resistance to PCT development were determined. PCT formation takes place over an extended period of 365 days but begins morphologically in focal proliferations of atypical plasma cells (foci) in the reactive oil granuloma that forms on mesenteric surfaces. Cells from some of these foci spread to other locations in oil granuloma tissue, forming new foci. Mice that develop six or more foci appear to be progressing towards eventual overgrowth and replacement of all peritoneal tissues with PCT cells. From Days 100 to 250, between 28 and 56% of PCT-susceptible BALB/cAn mice had 6 or more foci, whereas less than 5% of resistant DBA/2, BALB/c x DBA/2 F1 (hereafter called CD2F1), C57BL/6, and BALB/cJ mice had 6 or more foci. Four CD2 congenic strains carrying D2 alleles of genes on chromosomes other than chromosome 4 were highly susceptible. Between 0 and 20% of the mice in C.D2-Chr 4 congenic strains C.D2-MIA, C.D2-TF3, C.D2-Fv-1n/n, C.D2-Pnd7, C.D2-Lgm-1A, C.D2-Lgm-1B, C.D2-Lgm-1C, and C.D2-Lgm-1H developed 6 or more foci from 125 to 260 days, indicating resistance. The segments of DBA/2 chromosome 4 chromatin in C.D2-Fv-1n/n and C.D2-Pnd7 were discontinuous with those in C.D2-TF3, C.D2-Lgm-1A, C.D2-Lgm-1B, C.D2-Lgm-1C, and C.D2-Lgm-1H, indicating there are at least two genes (Pctr1 and Pctr2) in the distal half of this chromosome that confer resistance. Pctr1 is located between Ifa and D4Rck41, and Pctr2 is between Tnfr-1 and Pkcz. Each locus acting alone distinctly conferred a partial resistant phenotype. Pctr1 and Pctr2 did not appear to prevent the formation of clonal foci but did appear to limit the ability of the plasma cells in foci to acquire greater autonomy; thus, these genes affect tumor progression.

Plasmacytomas (PCTs) were induced in 47% of BALB/cAnPt mice by the intraperitoneal injection of pristane, in 2% of (BALB/c x DBA/2N)F1, and in 11% of 773 BALB/cAnPt x (BALB/cAnPt x DBA/2N)F1 N2 backcross mice. This result indicates a multigenic mode of inheritance for PCT susceptibility. To locate genes controlling this complex genetic trait, tumor susceptibility in backcross progeny generated from BALB/c and DBA/2N (resistant) mice was correlated with alleles of 83 marker loci. The genotypes of the PCT-susceptible progeny displayed an excess homozygosity for BALB/c alleles within a 32-centimorgan stretch of mouse chromosome 4 (> 95% probability of linkage) with minimal recombination (12%) near Gt10. Another susceptibility gene on mouse chromosome 1 may be linked to Fcgr2 (90% probability of linkage); there were excess heterozygotes for Fcgr2 among the susceptible progeny and excess homozygotes among the resistant progeny. Regions of mouse chromosomes 4 and 1 that are correlated with PCT susceptibility share extensive linkage homology with regions of human chromosome 1 that have been associated with cytogenetic abnormalities in multiple myeloma and lymphoid, breast, and endocrine tumors.

Many plasmacytomas arising in BALB/c mice are dependent upon a specific, macrophage-derived plasmacytoma growth factor for survival and proliferation in vitro. Adherent cells taken from the peritoneal oil granuloma in which the early plasmacytomas arise and proliferate produce 50 times more PCT-GF activity in vitro than do normal peritoneal cells, thus suggesting a possible in vivo role for PCT-GF. Purification and amino acid sequencing of PCT-GF derived from the murine macrophage cell line, P388D1, have identified a 23 kDa protein with a unique NH2-terminal sequence. This molecule is now known as murine IL6. As part of the characterization of murine Il-6, genomic sequences have been localized to the proximal end of mouse chromosome 5 via Southern analysis of restriction fragment length polymorphisms. The removal of IL6 from IL6-dependent PCT cell lines results in a growth arrest in early G1. This is accompanied by a rapid and specific loss of transferrin receptor expression and results in eventual cell death. It appears that the response to IL6 is at least partially dependent on Ca++ because functional Ca++ channels are necessary for the PCT cells to pass through G1 and to maintain transferrin receptor expression.