DZNep delays the engrafment and impairs the growth of MM cells in NSG mice.

<p>(<b>a</b>) RPMI 8226-GFP-Luc cells were injected into the caudal vein of NSG mice (n = 10) at day 1 (5×10⁶ cells were injected per animal). Three days later, mice were separated into two groups (n = 5 in each group), one received vehicle for control; the other was treated with 100 µg DZNEp twice a week as indicated in the scheme. At day 48, mice (except two, see below) were euthanized and tumors in soft tissues and bones removed for HES and IHC analyses. (<b>b</b>) BLI of the dorsal (D) and the ventral (V) sides of mice were taken at four sequential time points from day 3 to day 45. Both ventral and dorsal images of two mice in each group (mice #491/493 and #544/545) are shown. Mice 492 and 493 (red cross) showing hind leg paralysis were killed at day 36. (<b>c</b>) The luciferase activity of RPMI 8226-GFP-Luc cells in vehicle- (blue curves) and DZNep-treated (red curves) mice were determined into the two groups by BLI at the dorsal (plain line) and ventral (dotted line) levels.</p

EZH2 expression is reduced at post-transcriptional level but is not involved in MM cells response to DZNep treatment.

<p>(<b>a</b>) MMCLs were treated with either vehicle (0) or indicated concentrations of DZNep for 72 h. Western blots were performed with indicated antibodies. Anti-GAPDH Ab was used for loading and transfer control. The experiment has been repeated three times. (<b>b</b>) Responsive 8226 and resistant LP1 cell lines were treated for different time intervals (<b>upper part</b>) or 72 h (<b>lower part</b>). The transcriptional expression of <i>EZH2</i> (<b>upper part</b>) or <i>BMI1</i>, <i>EED</i> and <i>SUZ12</i> (<b>lower part</b>) was studied by qRT-PCR using the ΔΔC_t method with <i>RPLP0</i> as internal standard. The fold change was calculated as 2^{−ΔΔ<i>Ct</i>}. Indicated values corresponded to the mean ± SD from at least three independent experiments. (<b>c</b>) The responsive 8226 and JJN3 and the resistant LP1 cells were treated with vehicle, MG-132 (100 nM for 24 h), DZNep (1 µM for 24 h) or both. Cell were harvested; total proteins were purified, separated by SDS-PAGE and analyzed by Western blot with the indicated Abs. (<b>d</b>) 8226 cells were treated with CHX (100 µM for 1 h) then with vehicle or DZNep (1 µM for 24 h). Total proteins were purified, separated by SDS-PAGE and analyzed by Western blot with the indicated Abs.</p

DZNep-induced cell death is not necroptosis.

<p>(<b>a</b>) Responsive 8226 and resistant LP1 cells were either treated with vehicle or DZNep (1 µM for 24 h) then examined by transmission electronic microscopy. (<b>b</b>) Responsive 8226 and resistant LP1 cells were either treated with vehicle or DZNep (1 µM for 24 h). The perimeter (in µm), the area (in µm²) and the circularity (in arbitrary unit) of vehicle-(in white) or DZNep-treated (in black) LP1 and 8226 cells were determined by image analysis as described in the method section. (<b>c</b>) L363 cells were treated with vehicle, DZNep 1 µM for 24 h, or hydrogen peroxide (H₂O₂), incubated with CM-H₂DCFDA before FACS analysis. At least, 2×10⁴ events were gated. The percentage of stained cells (<i>i.e</i>. cells producing ROS) is indicated on the graph. (<b>d</b>) Responsive 8226 and resistant LP1 cell lines were treated for 6 or 24 h with DZNep 1 µM. The transcriptional expression of <i>TXNIP</i> was determined by qRT-PCR using the ΔΔC_t method with <i>RPLP0</i> as internal standard. The fold change was calculated as 2^{−ΔΔ<i>Ct</i>}. Indicated values corresponded to the mean ± SD from at least three independent experiments done with two distinct RNA preparations.</p

DZNep impairs the growth of MM cells in their niche.

<p>(<b>a</b>) Bones from control mice were processed and analyzed by HES and by IHC for CD138 and cleaved (cl.) caspase 3 staining. Images (x100 magnification) obtained for the femur of mouse #494 and the rachis of mouse #491, both vehicle-treated. CD138-positive cells that invaded bone marrow, caused bone (b) disorganization within the femur (#494) and the intervertebral discs (#491). Concomitantly, few cl. caspase 3 staining was noticed. (<b>b</b>) The femur (F) and tibia (T) from vehicle-treated mouse #517 were processed, scanned (x30 magnification), HE stained and analyzed for CD138 labeling by IHC (x100 magnification, a1, a2, b1; x630 magnification, a’1, b’1). Foci (a, b, c, circled regions) of typical CD138-membrane stained (b’1) MM cells tumors cells (tc) are visible within disorganized mouse bone marrow (m). MM cells mainly concentrated in trabecular areas (b, b1, b’1) but also in delineated medullae foci (a, a1, a’1, a2). (<b>c</b>) Examples of histological analyses (HES, CD138 and cl. caspase 3 staining) from rachis (mice #544 and 546), femur (mouse #548) and skull (mouse #545) samples removed from DZNep-treated series. CD138-positive <i>bona fide</i> MM cells invaded bone (b) tissues causing destruction and disorganization (puddles or ghosts associated with elevated caspase 3 activity). This high caspase 3 activity underlined DZNep therapeutic efficacy. Images of negative isotype rabbit or mouse IgG controls done on skull sections from mouse 454 are shown.</p

DZNep-induced cell death is apoptosis.

<p>(<b>a</b>) Exponentially growing MM cells were either treated with vehicle (DMSO 0.1%) or DZNep 1 µM for 72 h. Apoptosis was analyzed by FACS after APO2.7 staining. At least, 2×10⁴ events were gated. The percentage of apoptotic cells (stained for APO2.7) is indicated on the graph. (<b>b</b>) Exponentially growing JJN3 and 8226 cells were either pretreated with vehicle or Q-VD-OPh 10 µM for 1 h then with vehicle or DZNep 1 µM for 48 h. Cell proliferation was estimated by a MTS assay. Control samples referred to 100%. Here is shown a representative example from three independent experiments; each culture condition being in triplicate. Histograms show mean ± SD, *<i>p</i><0.05. (<b>c</b>) 8226, JJN3 and LP1 cells were treated with vehicle or Q-VD-OPh (10 µM for 1 h) and/or treated with DZNep (1 µM for 72 h). Cells were then stained with anti-APO2.7 Ab and analyzed by FACS. At least, 2×10⁴ events were gated. The percentage of apoptotic cells (stained for APO2.7) is indicated on the graph. (<b>d</b>) Western blots were performed with the indicated antibodies to study the caspase cascade; β-actin Ab was used as control of charge and transfer. White arrows show proforms of PARP and caspase 3/9 and black arrows the cleaved (cl.) and activated forms of proteins.</p

Localization of tumoral foci in NSG mice.

<p>The distribution of tumoral cells was assessed by BLI in ten mice (five per group), 45 days after the inoculation of 5×10⁶ RPMI 8226-GFP-Luc cells in the caudal vein of immunodeficient NSG mice.</p><p>Localization of tumoral foci in NSG mice.</p

Additivity of DZNep and bortezomib proapoptotic effects on MM cells.

<p>JJN3 DZNep-sensitive cells and LP1 DZNep-resistant cells were either treated with vehicle or DZNep 1 µM or bortezomib (Bort) 10–25 nM for 48 h or sequentially first with bortezomib for 24 h then DZnep for 24 h. Apoptosis was estimated after anti-APO2.7 staining of cells and cytometry sorting. At least, 2×10⁴ events were gated.</p><p>Additivity of DZNep and bortezomib proapoptotic effects on MM cells.</p

DZNep kills CD138⁺ MM cells and co-operates <i>in vitro</i> and <i>in vivo</i> with bortezomib.

<p>(<b>a</b>) Stromal HS-5 cells were seeded in 96-well plates at the density of 10⁴ cells/well and cultured for three days. Vehicle or DZNep (1 µM) was further added in five wells per culture condition and plates incubated for 24 and 48 h. Cell viability was determined by a MTS assay. On the graph are the means and SD values. ns, not significant. (<b>b</b>) Exponentially growing 8226 cells were separated into CD138^low and CD138^high populations. The sensitivity of CD138^high population and global population to DZNep (1 µM for 72 h) was compared by the MTS assay as before. ns, not significant with the Student’s <i>t</i>-test. (<b>c</b>) LP1 DZNep-resistant cells and JJN3 -sensitive cells were either treated with vehicle or DZNep 1 µM or bortezomib (Bort) 10–25 nM for 24 h or sequentially first with Bort then DZnep for 48 h. Cell proliferation was estimated by the MTS assay. Control samples referred to 100%. Here is shown a representative example from three independent experiments; each culture condition being in triplicate. Histograms show means ± SD, *<i>p</i><0.05.</p

DZNep co-operates <i>in vivo</i> with bortezomib.

<p>(<b>a</b>) RPMI 8226-GFP-Luc cells were injected into the caudal vein of NSG mice (n = 20) at day 1 (5×10⁶ cells were injected per animal). Ten days later, mice were separated into four groups (n = 5 in each group), one received vehicle for control, one was treated with 12.5 µg bortezomib i.p. twice a week, one was treated with 50 µg DZNep i.p. every two days, one was treated with bortezomib plus 50 µg DZNep i.p. every two days. Mice were imaged at days 15, 20, 32 and 39. At day 40, all mice (except three, see below) were euthanized. Two mice in the bortezomib-treated group and one in the bortezomib plus DZNep group died at day 32. BLI of the dorsal and the ventral sides of mice was taken at these time points and added. The luciferase activity (in arbitrary unit) representative of tumor growth in each series (mean ± SD) is represented in the graph; in blue the control group, in red the DZNep-treated group, in green the bortezomib-treated group and in purple the bortezomib/DZNep group. *<i>p</i><0.05 with the Student’s <i>t</i>-test. (<b>b</b>) Examples of histological analyses (HES, CD138 and cl. caspase 3 staining) from rachis (mouse #27955 from control group and mouse #745 from DZNep/borezomib group), left femur (mouse #827) from DZNep-treated group, and right femur (mouse #780) from bortezomib group. CD138-positive MM cells invaded bone tissues causing destruction and disorganization. In DZNep- or bortezomib-treated animals a high caspase 3 activity underlined therapeutic efficacy. Impressively, in mice #745 treated by both compounds, little CD138-positive cells were detected suggesting a possible cure.</p

DZNep induces myeloma cells death.

<p>Exponentially growing MM cells were either treated with vehicle (DMSO 0.1%) or DZNep 1 µM for 72 h (<b>a left part</b>) or 48–144 h (<b>a right part</b>). (<b>a</b>) Cell viability was assessed by Trypan blue exclusion. The percentage of viable cells referred to control experiments assigned to 100%. The experiment has been repeated three times, histograms show means ± SD, *<i>p</i><0.05. (<b>b</b>) Responsive 8226 and resistant LP1 cells were either treated with vehicle or DZNep (1 µM for 24 h) then examined by transmission electronic microscopy. (<b>c</b>) RPMI 8226 cells were treated with vehicle, the PI3K inhibitor LY94002 (1 µM for 24 h, <b>left part of the figure</b>), the mTOR inhibitor everolimus (10 nM for 24 h, <b>right part of the figure</b>), DZNep (1 µM for 24 h) or both and cell proliferation assayed by a MTS assay. For each culture condition, cells were seeded in three to five wells. The experiments have been repeated two or three times. A representative experiment is shown with the mean and SD values. ns, not significant.</p