Deletion of chromosomal region 8p21 confers resistance to Bortezomib and is associated with upregulated Decoy trail receptor expression in patients with multiple myeloma

Loss of the chromosomal region 8p21 negatively effects survival in patients with multiple myeloma (MM) that undergo autologous stem cell transplantation (ASCT). In this study, we aimed to identify the immunological and molecular consequences of del(8)(p21) with regards to treatment response and bortezomib resistance. In patients receiving bortezomib as a single first line agent without any high-dose therapy, we have observed that patients with del(8)(p21) responded poorly to bortezomib with 50% showing no response while patients without the deletion had a response rate of 90%. In vitro analysis revealed a higher resistance to bortezomib possibly due to an altered gene expression profile caused by del(8)(p21) including genes such as TRAIL-R4, CCDC25, RHOBTB2, PTK2B, SCARA3, MYC, BCL2 and TP53. Furthermore, while bortezomib sensitized MM cells without del(8)(p21) to TRAIL/APO2L mediated apoptosis, in cells with del(8)(p21) bortezomib failed to upregulate the pro-apoptotic death receptors TRAIL-R1 and TRAIL-R2 which are located on the 8p21 region. Also expressing higher levels of the decoy death receptor TRAIL-R4, these cells were largely resistant to TRAIL/APO2L mediated apoptosis. Corroborating the clinical outcome of the patients, our data provides a potential explanation regarding the poor response of MM patients with del(8)(p21) to bortezomib treatment. Furthermore, our clinical analysis suggests that including immunomodulatory agents such as Lenalidomide in the treatment regimen may help to overcome this negative effect, providing an alternative consideration in treatment planning of MM patients with del(8)(p21)

Quantitative RT-PCR analysis of gene expression in MM patients with or without del(8)(p21).

<p>TLDA cards were used to analyze mRNA levels in patients with (n = 19) and without (n = 6) del(8)(p21). For the analysis, GAPDH and ACTB genes were used as endogenous controls while RNA samples from K562 and U266 cell lines were used as calibrators. (*p<0.05, unpaired t test) (A) The mRNA level relative expression of TRAIL receptors in patients with and without the deletion. TRAIL-R1,-R2 and–R3 did not show any change while TRAIL-R4 was expressed at significantly higher levels in patients carrying the deletion. (B) Genes on or near 8p21 <b>(C)</b> other analyzed genes located elsewhere in the chromosome that show at least two-fold differential expression in MM cells with the deletion. Depicted are the gene expression levels in MM cells carrying the deletion, normalized to samples without the deletion.</p

Patients with del(8)(p21) fail to upregulate pro-apoptotic TRAIL receptor expression upon bortezomib treatment.

<p>Relative expression of TRAIL receptors of 5 MM patients with del(8)(p21) and 7 MM patients without deletion is determined by flow cytometry. Cell surface Expression levels are normalized to corresponding isotype controls. <b>(A)</b> TRAIL-R1, <b>(B)</b> TRAIL-R2, <b>(C)</b> TRAIL-R3, <b>(D)</b> TRAIL-R4. Effect of bortezomib treatment on TRAIL receptor expression is analyzed by paired t-test.</p

Patients with 8p21 deletion respond poorly to bortezomib treatment.

<p>Patients diagnosed with MM and receiving bortezomib based treatment at 1^st line were selected to this study (n = 88). (<b>A)</b> left panel is the non-HDT patients that received bortezomib (Vel: Velcade®, n = 33) as 1^st line treatment and right panel is the non-HDT patients that did not respond to 1^st line bortezomib treatment or relapsed and treated with Lenalidomide based regimen as a 2^nd line treatment (n = 16). B. Left panel displays patients received high dose treatment and bortezomib as 1^st line treatment (n = 55) and right panel is the response to Lenalidomide based treatment as 2^nd line for relapse patients (n = 18). Details of the treatments are shown in materials & methods section. Chi-square test was performed to analyze results.</p

Multivariate analysis of prognostic factors in multiple myeloma patients.

<p>Multivariate analysis of prognostic factors in multiple myeloma patients.</p

Multiple myeloma patients with del(8)(p21) have poor TTP and OS.

<p>Time to progression (TTP) of (A) all patients, (B) HDT patients, (C) non-HDT patients after diagnosis (months). Overall survival (OS) of (D) all patients, (E) HDT patients, (F) non-HDT patients after diagnosis (months). Patients with del(8)(p21) (n = 24). Patients without del(8)(p21) (n = 64). HDT patients with del(8)(p21) (n = 14). HDT patients without del(8)(p21) (n = 41). Non-HDT patients with del(8)(p21) (n = 10). Non-HDT patients without del(8)(p21) (n = 23). Log-rank (Mantel-cox) test performed using Graphpad Prism software.</p