HIF-1α and rapamycin act as gerosuppressant in multiple myeloma cells upon genotoxic stress

<p>Multiple myeloma (MM) is still an incurable hematological malignancy. Despite recent progress due to new anti-myeloma agents, the pathology is characterized by a high frequency of <i>de novo</i> or acquired resistance. Delineating the mechanisms of MM resistance is essential for therapeutic advances. We previously showed that long-term genotoxic stress induces the establishment of a senescence-associated secretory phenotype, a pro-inflammatory response that favors the emergence of cells with cancer stem-like properties. Here, we studied the short-term response of MM cells following treatment with various DNA damaging agents such as the energetic C-ion irradiation. MM cells are highly resistant to all treatments and do not enter apoptosis after they arrest cycling at the G2 phase. Although the DNA damage response pathway was activated, DNA breaks remained chronically in damaged MM cells. We found, using a transcriptomic approach that <i>RAD50</i>, a major DNA repair gene was downregulated early after genotoxic stress. In two gerosuppression situations: induction of hypoxia and inhibition of the mammalian target of rapamycin (mTOR) pathway, we observed, after the treatment with a DNA damaging agent, a normalization of RAD50 expression concomitant with the absence of cell cycle arrest. We propose that combining inhibitors of mTOR with genotoxic agents could avoid MM cells to senesce and secrete pro-inflammatory factors responsible for cancer stem-like cell emergence and, in turn, relapse of MM patients.</p

MM mouse model.

<p>(A) Sequential BLI scans of a mouse injected with 2 x 10⁶ RPMI 8226-GFP-Luc cells into the flank. (B) Luciferase activity quantified at the same time points for ten mice, mean ± SD. (C) Representative immunohistochemistry images (x100 magnification) of tumour sections of the mouse, the BLI scans of which are displayed above. HES: nuclear stain, CD138: identity of the cancer cells-of-origin, p-H3: reflects proliferation, CD34: reflects neoangiogenesis.</p

Combined PET/CT and BLI data.

<p>(A) Representative [¹⁸F]fludarabine- and (B) [¹⁸F]FDG-PET/CT fused scans (40–60 min post-injection), as well as (C) the corresponding BLI scan from the same mouse. (D) Relationship between CT-based tumour volume (mean ± SD, in duplicate) and BLI intensity. (E) Relationship between [¹⁸F]-labelled tracers uptake and BLI intensity.</p

<i>In vitro</i> analysis of BLI and [¹⁸F]fludarabine signals.

<p>Relationship between the number of RPMI8226-GFP-Luc cells and (A) BLI intensity, (B) [¹⁸F]fludarabine uptake. Error bar: mean ± SD, in triplicate.</p

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

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 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

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 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