The major new finding reported here is that the effect of PI3K/mTOR blockade on cell cycle progression in MM cells is critically dependent on the underlying mode of cyclin D dysregulation, which in turn is dictated by IgH translocation status. The PI3K pathway is a major downstream signalling pathway utilised by the most important growth/survival factors for MM. Such factors, thought to have a role in mediating the protective effect of the myeloma BM microenvironment include IGF-I, IL-6, vascular endothelial growth factor, fibroblast growth factor, hepatocyte growth factor, APRIL and BAFF.22, 23
We and others have shown previously that in addition to its well-characterised role in mediating cell survival, PI3K is essential for mediating cell cycle entry and expression of D-type cyclins in B-lineage cells. Notably, mice that are genetically deficient in Class I PI3K are deficient in B-cell development and activation, and are unable to induce cyclin D2 normally in response to mitogens.11, 24
Furthermore, PI3K-knockout mice are phenotypically similar to mice lacking cyclin D2.11, 25
Using a panel of HMCL with different IgH translocations, we observed that cells bearing t(4;14) or t(14;16) were more sensitive to PI-103-mediated growth inhibition when compared with t(11;14)-bearing cells. This differential sensitivity to PI-103 was apparent when cells were grown in their standard 10% FCS medium or in IGF-I alone. Further analysis revealed that these differences are largely due to differential effects on the cell cycle, with PI-103 treatment leading to cell cycle arrest in the t(4;14)- or t(14;16)-bearing cells, but not in t(11;14)-bearing cells. The cell cycle arrest observed in t(4;14) and t(14;16) cells was mediated by a decrease in cyclin D2, CDK 4 and CDK6 expression and an increase in levels of p27, concomitant with decreased CDK4/6-specific pRb phosphorylation and total pRb. These changes are similar to those observed in normal B lymphocytes undergoing cell cycle arrest due to serum deprivation or PI3K inhibition,24, 26, 27
where decreased cyclin D2-CDK4/6 complexes are unable to titrate p27 (a CIP/WAF family CKI) away from cyclin E/CDK2 complexes, leading to further dephosphorylation of pRb and resultant repression of the E2F transcription factor. Total pRb is also downregulated as it is itself an E2F-regulated gene. In contrast to cyclin D2-expressing cells, and in keeping with the lack of cell cycle inhibition, there were little or no changes observed in G1-phase cell cycle control proteins in t(11;14) cells treated with PI-103.
Here, we show, for the first time, that PI3K inhibition modulates cyclin D2 protein levels in primary CD138+ MM cells. We also confirm, in a larger cohort of patients, our earlier findings that, in primary MM cells bearing t(4;14) or t(14;16), cyclin D2 is functionally upregulated by IGF-1, unlike cyclin D1 expressed via a t(11;14), which is unresponsive to IGF-1. IGF-I stimulation increased protein levels of cyclin D2 as well as CDK4/6-specific pRb phosphorylation in MM cells from patients harbouring t(4;14) or t(14;16). We have recently reported that APRIL modulates cyclin D2 and cell cycle proteins to induce cell cycle progression in t(4;14)- and t(14;16)-bearing primary MM cells.8
Such cell cycle responses to APRIL were absent in cyclin D1MM cells. In t(4;14) or t(14;16) cells, cyclin D2 is upregulated indirectly by overexpression of the MMSET/FGFR3 proteins or by the c-MAF transcription factors, which may themselves be subjected to regulation by mitogens. Thus, it is unsurprising that cyclin D2 which is under the control of its natural, growth-factor responsive promoter is sensitive to PI3K inhibition, because PI3K is known to be a major regulator of this protein in haematopoietic cells. MM cells carrying a t(11;14), however, express cyclin D1 constitutively by the strong IgH enhancer elements, thus rendering it independent of mitogenic signalling. What is perhaps surprising is that other cell cycle proteins, such as CDK4/6, total pRb and p27 are also unaffected by mitogens and/or PI3K inhibition in t(11;14) cells, because expression of these proteins should remain under normal physiological regulation. This is especially pertinent for p27, which is known to be regulated by PI3K.28, 29
It is possible that in the presence of excess cyclin D1, the cyclin D1-CDK4/6-p27 complexes are stably maintained and/or exert feedback signalling that overrides regulation by mitogenic withdrawal or PI3K inhibition. Alternatively, t(11;14) MM cells may evolve in a mitogen-independent manner, so that upregulation of cyclin D1 (an early, premalignant event) is associated with later mutations that maintain constitutive expression of CDK4/6-p27 complexes independently of PI3K activity. However, because most primary t(11;14) MM cells are not constitutively in cycle,30
there must be as yet unrecognised signals that regulate their cell cycle entry in vivo
Although this study has focussed on MM cells harbouring an IgH translocation, approximately 50% of MM cases are characterised by hyperdiploidy and an absence of an IgH translocation. It is technically challenging to investigate the mechanisms of cell cycle control in this group, because they are not adequately represented by HMCL. However, recent data from our laboratory, utilising an improved culture method for primary MM cells, have demonstrated that in the hyperdiploid group, cyclin D2-expressing tumours are more sensitive to growth factors than cyclin D1-expressing tumours, but are less responsive when compared with cyclin D2-expressing cases harbouring t(14;16) or t(4;14).8
This study has focussed on cell cycle regulation, however, other work from our group has shown that dependence on survival signalling from the PI3K pathway is also dictated by IgH translocation status.31
These data support the hypothesis that t(11;14) cells clonally evolve in a manner that makes them relatively refractory to signals emanating from these classical myeloma growth/survival factors when compared with those harbouring IgH translocations targeting cyclin D2.
The relative insensitivity of t(11;14) cells to PI-103 could be explained by a functionally inactive PI3K pathway. Alternatively, they might have a hyper-activated pathway (for example, due to mutation of PI3K subunits) refractory to enzymatic inhibition. Neither of these explanations, however, is borne out by our observations on the activation of key PI3K signalling intermediates. When we compared MM cells representing the three major IgH translocation groups, we found no differences in IGF-I-induced activation of PKB. Furthermore, PI-103 abrogation of PKB phosphorylation was equally apparent in t(11;14) cells compared with t(4;14) or t(14;16) cells. Thus, the differential impact of PI3K/mTOR blockade on cell cycle progression between cyclin D2-expressing t(4;14) or t(14;16) and cyclin D1-expressing t(11;14) MM tumours may be due to the composition and activity of cyclin D—CDK complexes, and their regulation by proteolytic mechanisms. Differential sensitivity to PKB inhibition has been observed by Zollinger et al.,32
who attributed this to differences in constitutive PKB phosphorylation both in HMCL and primary MM cells. Although the emphasis was on apoptosis rather than cell cycle regulation, the HMCL shown to be sensitive to PKB inhibition were OPM2 and MM1S, which are t(4;14) and t(14;16) positive, respectively, whereas the t(11;14) HMCL, U266 was insensitive. These, and our findings reported here, raise the possibility that IgH/cyclin D status may dictate patients' response to therapies aimed at inhibition of PKB and other targets downstream of PI3K. Several genetic abnormalities have been associated with increased PKB activation, including mono-allelic deletion of PTEN, present in approximately 5% of BM MM cells, and up to 20% in plasma cell leukaemias and HMCL.33
However, PTEN mutations were found to be absent in most MM cells with constitutively activated PKB, while ‘hotspot' mutations in PIK3CA (which codes for the p110 alpha catalytic subunit of PI3K) and PKB identified in a variety of other tumours were not found in MM,34
suggesting either that other genetic abnormalities are responsible or that the activation is due to increased growth factor responsiveness.
We used primary MM cells to test whether the inhibitory effect of PI-103 on cell cycle progression would enhance the activity of anti-MM agents. We observed, in primary t(14;16)-bearing MM cells, that the addition of PI-103 to melphalan, bortezomib, dexamethasone or lenalidomide resulted in a greater inhibition of DNA synthesis and greater reduction in levels of cyclin D2 and CDK4-/6-phosphorylated pRb than observed with either drug alone. In contrast, in t(11;14)-bearing cells, no such potentiating effect was observed. These data indicate that combination therapies utilising PI3K/mTOR inhibitors would be particularly effective in those patients with IgH translocations that target cyclin D2. The importance of dual inhibition of both PI3K and mTOR pathways has been recently recognized.14
Although PI-103 itself is unlikely to progress to clinical development because of its limited solubility and extensive metabolism, several pharmacologically optimised PI3K/mTOR inhibitors are in development. One such agent, NVP-BEZ235, was also recently shown to inhibit MM cell growth and proliferation and exhibit synergy with anti-MM drugs in two independent studies.16, 35
McMillin et al.
in their work on HMCL, noted that there was varying sensitivity to the effect of this PI3K/mTOR inhibitor, but did not report any correlation with genetic lesion. Using NVP-BEZ235, we demonstrate that dual blockade of PI3K/mTOR downregulates cyclin D2 and CDK6 in vivo
, leading to an attenuation of tumour growth in MM cells bearing t(4;14).
In summary, we report that the effect of PI3K/mTOR inhibition on cell cycle progression in MM cells segregates with IgH/D-type cyclin status. Importantly, tumour cells from patient subgroups with inferior outcomes, and characterised by higher proliferative behaviour, including responses to mitogens, are particularly sensitive to PI3K pathway blockade. These tumour cells express cyclin D2 in conjunction with the t(4;14) or t(14;16), whereas tumours with a different genetic background, that is, expressing cyclin D1 with t(11;14), that confers a neutral prognosis, are largely insensitive. Our findings provide the rationale for the incorporation of dual PI3K/mTOR inhibitors in treatment strategies aimed at patients with t(4;14) or t(14;16), and set the scene for future mechanistic studies on cell cycle regulation in these distinct subtypes of MM.