FGFR3 preferentially colocalizes with IGH in the interphase nucleus of multiple myeloma patient B-cells when FGFR3 is located outside of CT4.

Many B-cell malignancies are characterized by chromosomal translocations involving IGH and a proto-oncogene. For translocations to occur, spatial proximity of translocation-prone genes is necessary. Currently, it is not known how such genes are brought into proximity with one another. Although decondensed chromosomes occupy definitive, non-random spaces in the interphase nucleus known as chromosome territories (CTs), chromatin at the edges of CTs can intermingle, and specific genomic regions from some chromosomes have been shown to "loop out" of their respective CTs. This extra-territorial positioning of specific genomic regions may provide a mechanism whereby translocation-prone genes are brought together in the interphase nucleus. FGFR3 and MAF recurrently participate in translocations with IGH at different frequencies. Using 3D, 4-color FISH, and 3D analysis software, we show frequent extra-territorial positioning of FGFR3 and significantly less frequent extra-territorial positioning of MAF. Frequent extra-territorial positioning may be characteristic of FGFR3 in B-cells from healthy adult donors and non-malignant B-cells from patients, but not in hematopoietic stem cells from patients with translocations. The frequency of extra-territorial positioning of FGFR3 and MAF in B-cells correlates with the frequency of translocations in the patient population. Most importantly, in patient B-cells, we demonstrate a significant proportion of extra-territorial FGFR3 participating in close loci pairs and/or colocalizing with IGH. This preliminary work suggests that in patient B-cells, extra-territorial positioning of FGFR3 may provide a mechanism for forming close loci pairs and/or colocalization with IGH; indirectly facilitating translocation events involving these two genes. © 2016 Wiley Periodicals, Inc.

Differential nuclear organization of translocation-prone genes in nonmalignant B cells from patients with t(14;16) as compared with t(4;14) or t(11;14) myeloma.

Gene organization in nonmalignant B cells from t(4;14) and t(11;14) multiple myeloma (MM) patients differs from that of healthy donors. Among recurrent IGH translocations in MM, the frequency of t(4;14) (IGH and FGFR3) or t(11;14) (IGH and CCND1) is greater than the frequency of t(14;16) (IGH and MAF). Gene organization in t(14;16) patients may influence translocation potential of MAF with IGH. In patients, three-dimensional FISH revealed the positions of IGH, CCND1, FGFR3, and MAF in nonmalignant B cells that are likely similar to those when MM first arose, compared with B cells from healthy donors. Overall, IGH occupies a more central nuclear position while MAF is more peripherally located. However, for B cells from t(4;14) and t(11;14) patients, IGH and FGFR3, or IGH and CCND1 are found in spatial proximity: IGH and MAF are not. This differs in B cells from t(14;16) patients and healthy donors where IGH is approximately equidistant to FGFR3, CCND1, and MAF, suggesting that gene organization in t(14;16) patients is different from that in t(4;14) or t(11;14) patients. Translocations between IGH and MAF may arise only in the absence of close proximity to the more frequent partners, as appears to be the case for individuals who develop t(14;16) MM.

Differential positioning and close spatial proximity of translocation-prone genes in nonmalignant B-cells from multiple myeloma patients.

Accumulating evidence suggests that spatial proximity of potential chromosomal translocation partners influences translocation probability. It is not known, however, whether genome organization differs in nonmalignant cells from patients as compared to their cellular counterparts from healthy donors. This could contribute to translocation potential causing cancer. Multiple myeloma is a hematopoietic cancer of the B-lineage, characterized by karyotypic instability, including chromosomal translocations involving the IGH locus and several translocation partners. Utilizing 3-D FISH and confocal imaging, we investigate whether nuclear spatial positioning of the translocation-prone gene loci, IGH, FGFR3, and CCND1 differs in nonmalignant cell subsets from multiple myeloma patients as compared to positioning in their corresponding healthy donor cell subsets. 3-D analysis software was used to determine the spatial proximity of potential translocation pairs and the radial distribution of each gene. We observed that in all cell subsets, the translocation-prone gene loci are intermediately located in the nucleus, while a control locus occupies a more peripheral position. In nonmalignant B-cells from multiple myeloma patients, however, the translocation-prone gene loci display a more central nuclear position and close spatial proximity. Our results demonstrate that gene positioning in nonmalignant B-cells from multiple myeloma patients differs from that in healthy donors, potentially contributing to translocation probability in patient cells. We speculate that genome reorganization in patient B-cells may closely reflect gene positioning at the time the multiple myeloma-specific translocation initially formed, thus influencing translocation probability between proximal loci in the B-cell population from which the malignancy emerged.

Promiscuity of translocation partners in multiple myeloma.

Multiple myeloma (MM) is characterized by karyotypic instability, including chromosomal translocations involving the IGH locus. MM cells display a promiscuity of translocation partners, only some of which are recurrent. We propose that several factors, including temporal and spatial nuclear positioning of potential partner loci, "off-target" IGH diversification mechanisms, and aberrant repair pathways contribute to the promiscuity of translocation partners in MM. We speculate that in MM, IGH diversification processes [V(D)J recombination, somatic hypermutation, and class switch recombination] in B cells may not be restricted to specific stages of B-cell development or within specific immune tissues, but may occur in different temporal "windows." Before or during MM evolution, off-target activities of the enzymes involved in IGH modification processes may contribute to the generation of double-strand breaks (DSB) in translocation partner loci. In the parent B cells from which MM originates, spatial proximity within the nucleus of IGH and potential translocation partners contributes to the selection of a translocation partner and the clinical frequency at which a specific translocation occurs. The spatial proximity of IGH and specific translocation partners may be temporal and contribute not only to partner selection but also to the promiscuity of partners seen in MM. Lastly, aberrant repair mechanisms in MM progenitors (including the possibility that a Ku 86 variant allows for positional instability at DSBs) may also contribute to the promiscuity of chromosome translocation partners in MM.

A unique three-dimensional model for evaluating the impact of therapy on multiple myeloma.

Although the in vitro expansion of the multiple myeloma (MM) clone has been unsuccessful, in a novel three-dimensional (3-D) culture model of reconstructed bone marrow (BM, n = 48) and mobilized blood autografts (n = 14) presented here, the entire MM clone proliferates and undergoes up to 17-fold expansion of malignant cells harboring the clonotypic IgH VDJ and characteristic chromosomal rearrangements. In this system, MM clone expands in a reconstructed microenvironment that is ideally suited for testing specificity of anti-MM therapeutics. In the 3-D model, melphalan and bortezomib had distinct targets, with melphalan targeting the hematopoietic, but not stromal com-partment. Bortezomib targeted only CD138(+)CD56(+) MM plasma cells. The localization of nonproliferating cells to the reconstructed endosteum, in contact with N-cadherin-positive stroma, suggested the presence of MM-cancer stem cells. These drug-resistant CD20(+) cells were enriched more than 10-fold by melphalan treatment, exhibited self-renewal, and generated clonotypic B and plasma cell progeny in colony forming unit assays. This is the first molecularly verified demonstration of proliferation in vitro by ex vivo MM cells. The 3-D culture provides a novel biologically relevant preclinical model for evaluating therapeutic vulnerabilities of all compartments of the MM clone, including presumptive drug-resistant MM stem cells.