Abstract 5080

Introduction:

Multiple myeloma (MM) cells depend on the bone marrow (BM) niche signals for their viability. Thus, tracing the earliest molecular changes in response to the lost of niche signals can disclose new homing related interactions.

Methods:

BM aspirates were collected in heparinized syringes and samples were flesh frozen at different time points. GEP (Affymetrix 1.0 st) was performed for 8 cases with massive BM infiltration with plasma cells. The GEP was also analyzed at different time points following spreading of cultured BM cells from the same patients (maintained in culture for several days).

Results:

Spontaneous induction of genes started within few minutes following aspiration, peaking in hours and persisting for at least 12 hours (Tables 1). RT-PCR results along several time points following BM aspiration from a representative case are presented in Table 2. Interestingly, the induction of genes reverted entirely in cultured conditions imposing cell-cell-matrix contact. However, spreading of the cultured cells re-induced these genes similar to that followed BM aspiration. Furthermore, in comparison of the GEP of cultured cells (frozen immediately after spreading) Vs the GEP from samples taken immediately after BM aspiration, an absolutely different repertoire of genes showed significant changes (Table 3):

Table 1:

Genes induced >120 minutes following BM aspiration

GeneRank*Average FC**
FOS 14.4 21.6 (6.5–38.1) 
FOSB 13.4 20.9 (7.5–40.3) 
RGS1 11.2 23.3 (5.2–87.4) 
MOP-1 11.1 14.2 (5.9–38.1) 
DUSP1 10.6 11.3 (4.1–28.2) 
AREG 9.1 24.8 (3.2–65.0) 
JUN 8.8 10.6 (3.1–29.4) 
NR4A2 5.6 8.7 (3.5–17.8) 
AREG 8.8 15.4 (2.1–48.1) 
CD69 6.9 5.6 (4.4–6.9) 
PTGS2 6.4 5.6 (2.8–11.3) 
EGR1 5.6 6.0 2.3–17.2) 
TRIB1 3.7 3.7 (2.0–5.0) 
OTUD1 2.6 4.3 (1.6–8.3) 
DUSP10 2.1 4.2 (1.6–8.3) 
CCL3 1.3 3.9 (1.4–10.4) 
PPP1R15A 1.0 3.4 (2.0–7.5) 
GeneRank*Average FC**
FOS 14.4 21.6 (6.5–38.1) 
FOSB 13.4 20.9 (7.5–40.3) 
RGS1 11.2 23.3 (5.2–87.4) 
MOP-1 11.1 14.2 (5.9–38.1) 
DUSP1 10.6 11.3 (4.1–28.2) 
AREG 9.1 24.8 (3.2–65.0) 
JUN 8.8 10.6 (3.1–29.4) 
NR4A2 5.6 8.7 (3.5–17.8) 
AREG 8.8 15.4 (2.1–48.1) 
CD69 6.9 5.6 (4.4–6.9) 
PTGS2 6.4 5.6 (2.8–11.3) 
EGR1 5.6 6.0 2.3–17.2) 
TRIB1 3.7 3.7 (2.0–5.0) 
OTUD1 2.6 4.3 (1.6–8.3) 
DUSP10 2.1 4.2 (1.6–8.3) 
CCL3 1.3 3.9 (1.4–10.4) 
PPP1R15A 1.0 3.4 (2.0–7.5) 
*

based on pooled data. The mildest inducible transcript was defined as 1.0;

**

FC=fold change

Table 2:

RT-PCR recording changes in the expression of selected genes over time

Time from aspirationFold change
3 min10 min17 min28 min40 min80 min120 min
FOS 1.2 4.9 37.7 112 223 309 277 
FOSB 0.9 1.3 3.7 186 2385 6393 9614 
MOP-1 1.67 3.4 9.4 17.7 32.8 24.3 23.5 
Time from aspirationFold change
3 min10 min17 min28 min40 min80 min120 min
FOS 1.2 4.9 37.7 112 223 309 277 
FOSB 0.9 1.3 3.7 186 2385 6393 9614 
MOP-1 1.67 3.4 9.4 17.7 32.8 24.3 23.5 
Table 3:

Comparison of the GEP in cultured primary MM cells vs fresh BM aspiration cells

UpregulatedDownregulated
Gene SymbolFCGene SymbolFCGene SymbolFC
IL8 22.3 PLCB1 2.1 CNTNAP3 3.2 
SPP1 22.1 C6orf167 2.2 MYB 3.2 
CXCL5 21.2 DEPDC1B 2.2 CD24 3.2 
SERPINB2 19.8 CRISPLD1 2.3 VCAM1 3.2 
ADAMDEC1 12.2 PLSCR4 2.4 CENPE 3.2 
CCL2 11.4 LOC643332 2.4 TOP2A 3.2 
PLA2G7 8.03 KIF18A 2.4 TUBB1 3.2 
FN1 7.97 KIF20B 2.4 DLGAP5 3.2 
FTH1P2 7.74 LOC100289612 2.5 CEACAM6 3.3 
MMP12 6.97 SYT1 2.5 CD200 3.3 
GPNMB 6.89 SKA3 2.5 MIR144 3.3 
CCL20 6.87 ITGA8 2.6 RGS5 3.3 
CTSL1 6.54 ART4 2.8 SLC25A37 3.4 
CYP1B1 6.23 TLR1 2.8 SGOL1 3.4 
DDIT4 5.58 SLC40A1 2.8 CENPF 3.4 
THBS1 4.94 TMOD1 2.8 CD36 3.4 
C3 4.93 CNTN1 2.8 SULT1B1 3.4 
ALDOC 4.84 MAL2 2.8 LRRK2 3.4 
MT1G 4.54 NUF2 2.8 SELL 3.5 
TGFBI 4.13 LCN2 2.8 RSAD2 3.5 
PLIN2 3.97 EXO1 2.9 ASPM 3.6 
C13orf31 3.96 CD5L 2.9 IFI44 3.7 
IL6 3.7 PCDH18 2.9 MGAM 3.7 
FTL 3.68 CA2 2.9 HEMGN 3.7 
CYP27A1 3.55 CASC5 2.9 ABCA13 3.8 
WNT5A 3.47 KIF14 2.9 SLFN14 3.8 
TMEM147 3.38 ESCO2 2.9 MMRN1 3.9 
MIR29A 3.33 KIF23 3.0 HBD 3.9 
HBEGF 3.21 SAMD9L 3.0 HIST1H2BM 4.0 
CA12 2.94 SELENBP1 3.0 S100A12 4.0 
CTSB 2.94 ARHGAP11A 3.0 CXCR1 4.1 
SELM 2.92 NEIL3 3.0 FGL2 4.2 
PRDX5 2.88 MKI67 3.0 P2RY13 4.2 
CD68 2.8 RNASE3 3.1 BPI 4.2 
SOCS3 2.65 DEPDC1 3.1 FAR2 4.3 
CTSD 2.56 HP 3.1 CXCR2 4.7 
LOC402778 2.17 XAF1 3.1 PLBD1 4.7 
  KIF20A 3.1 IFIT1B 5.2 
  KIF11 3.1 MS4A3 5.9 
  MANSC1 3.1 IFI44L 6.5 
UpregulatedDownregulated
Gene SymbolFCGene SymbolFCGene SymbolFC
IL8 22.3 PLCB1 2.1 CNTNAP3 3.2 
SPP1 22.1 C6orf167 2.2 MYB 3.2 
CXCL5 21.2 DEPDC1B 2.2 CD24 3.2 
SERPINB2 19.8 CRISPLD1 2.3 VCAM1 3.2 
ADAMDEC1 12.2 PLSCR4 2.4 CENPE 3.2 
CCL2 11.4 LOC643332 2.4 TOP2A 3.2 
PLA2G7 8.03 KIF18A 2.4 TUBB1 3.2 
FN1 7.97 KIF20B 2.4 DLGAP5 3.2 
FTH1P2 7.74 LOC100289612 2.5 CEACAM6 3.3 
MMP12 6.97 SYT1 2.5 CD200 3.3 
GPNMB 6.89 SKA3 2.5 MIR144 3.3 
CCL20 6.87 ITGA8 2.6 RGS5 3.3 
CTSL1 6.54 ART4 2.8 SLC25A37 3.4 
CYP1B1 6.23 TLR1 2.8 SGOL1 3.4 
DDIT4 5.58 SLC40A1 2.8 CENPF 3.4 
THBS1 4.94 TMOD1 2.8 CD36 3.4 
C3 4.93 CNTN1 2.8 SULT1B1 3.4 
ALDOC 4.84 MAL2 2.8 LRRK2 3.4 
MT1G 4.54 NUF2 2.8 SELL 3.5 
TGFBI 4.13 LCN2 2.8 RSAD2 3.5 
PLIN2 3.97 EXO1 2.9 ASPM 3.6 
C13orf31 3.96 CD5L 2.9 IFI44 3.7 
IL6 3.7 PCDH18 2.9 MGAM 3.7 
FTL 3.68 CA2 2.9 HEMGN 3.7 
CYP27A1 3.55 CASC5 2.9 ABCA13 3.8 
WNT5A 3.47 KIF14 2.9 SLFN14 3.8 
TMEM147 3.38 ESCO2 2.9 MMRN1 3.9 
MIR29A 3.33 KIF23 3.0 HBD 3.9 
HBEGF 3.21 SAMD9L 3.0 HIST1H2BM 4.0 
CA12 2.94 SELENBP1 3.0 S100A12 4.0 
CTSB 2.94 ARHGAP11A 3.0 CXCR1 4.1 
SELM 2.92 NEIL3 3.0 FGL2 4.2 
PRDX5 2.88 MKI67 3.0 P2RY13 4.2 
CD68 2.8 RNASE3 3.1 BPI 4.2 
SOCS3 2.65 DEPDC1 3.1 FAR2 4.3 
CTSD 2.56 HP 3.1 CXCR2 4.7 
LOC402778 2.17 XAF1 3.1 PLBD1 4.7 
  KIF20A 3.1 IFIT1B 5.2 
  KIF11 3.1 MS4A3 5.9 
  MANSC1 3.1 IFI44L 6.5 
Conclusions:

1. BM cells from MM patients respond immediately to changing microenvironment, with prompt upregulation of genes active in tumor expansion (FOS, AREG, JUN, PTGS2). However, such constitutive upregulation plus elevation of tumor suppressor genes (like NR4A2) is intolerable if not reverted by cell-cell-matrix contact. 2. In culture, BM cells from MM patients respond with dramatic upregulation of genes supporting neovascularisation (IL8, PLA27, MMP12, CXCL5), matrix (SPP1, FN1) and osteoclasts. 3. Most interestingly, in culture there is also downregulation of 20 genes critical to mitosis, especially relating to spindle machinery (KIF members, NUF2, TUBB1, ASPM, CENPE, CENPF) suggesting that the failure of long-term culture of primary MM cells results from mitotic crisis. This may also explain the lobulation of primary MM cell nuclei observed. 4. The unannotated gene MOP-1 has a role in MM homing.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.