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Stimulation of B lymphocytes through the Ig receptor initiates a cascade of biochemical changes, which can ultimately lead to either activation and growth, or cell-cycle arrest and cell death. One of the critical events that occurs in both cases is the activation of tyrosine kinases, and the resulting phosphorylation of a variety of proteins on tyrosine residues. In this report we identify one of the substrates of phosphorylation as the 85-kD subunit of the enzyme phosphatidylinositol-3 kinase (PI3K), and show that both anti-IgM and anti-IgD stimulation results in an increase in the anti-phosphotyrosine-precipitable PI3K activity. Furthermore, we show that the potent and specific inhibitor of PI3K, Wortmannin, can completely abrogate anti-Ig-mediated growth inhibition without affecting tyrosine kinase induction or protein kinase C (PKC) activation. Treatment of intact cells with Wortmannin results in an irreversible decrease in anti-Ig-induced PI3K activity, suggesting that the effect of Wortmannin on anti-Ig-mediated growth inhibition is caused by its inactivation of PI3K activity. Taken together, these data show that activation of PI3K is a critical component of the anti-Ig-initiated signaling cascade that leads to growth inhibition of human B lymphoma cells.

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells in the bone marrow caused primarily by failure of normal homeostatic mechanisms to prevent the expansion of postgerminal center plasma cells. We have examined the molecular mechanisms that promote the survival of MM cells and have identified a key role for myeloid cell factor–1 (Mcl-1), an antiapoptotic member of the Bcl-2 family. These experiments were initiated by the observation that MM cells were exquisitely sensitive to culture in the presence of actinomycin D: caspase activation occurred within 3 hours of treatment and cells were not protected by interleukin-6, the main MM cell growth and survival factor. Actinomycin D–induced apoptosis was blocked by proteasome inhibitors, suggesting that a labile protein was required for MM cell survival. Further analysis demonstrated that Mcl-1 was likely to be the labile factor governing MM cell survival. Mcl-1 protein levels decreased rapidly after culture in the presence of actinomycin D in concordance with effector caspase activation, but addition of proteasome inhibitors reversed the loss of Mcl-1 and maintained cell viability. The levels of other antiapoptotic proteins, including Bcl-2 and members of the inhibitors-of-apoptosis family, were unaffected by these interventions. Furthermore, Mcl-1 antisense oligonucleotides caused a rapid down-regulation of Mcl-1 protein levels and the coincident induction of apoptosis, whereas overexpression of Mcl-1 delayed actinomycin D–induced apoptosis with kinetics that correlated with expression levels of Mcl-1. These data indicate that Mcl-1 expression is required for the survival of MM cells and may represent an important target for future therapeutics.

Bortezomib has demonstrated activity in heavily pretreated MM patients. The dose limiting toxicity is peripheral neuropathy (PN) that affects up to 35% of the patients (Richardson et al JCO 2006). We retrospectively reviewed the incidence and severity of PN in 78 patients who received bortezomib at our institution. The median age was 57 years (range: 33–80), 62% were men, and 37% were African Americans (AA). Risk factors for PN included prior use of thalidomide in 53 patients (68%) and vincristine in 31 patients (40%). Seventeen patients (22%) had diabetes mellitus. Before bortezomib treatment 29 patients (37%) reported subjective grade 1–2 PN. Patients received bortezomib alone (n=10) or in combination with dexamethasone (n=36) and thalidomide (n=20) or chemotherapy (n=12). Responses included complete, near complete and partial responses in 5%, 10% and 27%, respectively. Grade 2 and higher PN occurred in 52% of the patients including Grade grades 3 and 4 PN in 15% and 7%, respectively. Twelve patients stopped bortezomib due to side effects that included PN (n=8), diarrhea (n=2) and CMV pneumonia (n=1), and 11 patients had dose reductions because of PN and fatigue. Grade 4 PN affected 6 patients (sensory n=4, and motor/sensory, n=2), 5 of whom were AA and 4 of whom had DM. The onset of grade 4 PN was acute rather than cumulative as noted with lower grades and took a median of 8 months (range: 4–16+) to improve compared to 3–4 months for lower grade PN. Neuropathy grade was not associated with age, sex, race or renal function (10 patients had creatinine > 2 mg/dl, including 2 on dialysis). Factors predictive of PN were baseline neuropathy (p=0.002) in addition to prior thalidomide use (p=0.03) and presence of DM (p=0.03). Responses were independent of neuropathy grade and whether bortezomib was combined with chemotherapy or thalidomide. The duration of therapy in responding patients was longer in patients receiving bortezomib in combination with thalidomide with a median of 5 cycles (range: 2–36) versus those who received other bortezomib combinations 3 cycles (range 1–19). Two of four patients with grade 4 sensory PN had their best response ever after 3 cycles of bortezomib; both had failed transplant and thalidomide based therapies; one remains in continuous complete remission for 24 months. PN therapy was mostly supportive including combinations of analgesics duloxetine, gabapentin, and pregabalin. Interestingly, 6 of 9 patients with PN who received lenalidomide as salvage therapy after progression on bortezomib had significant improvement in their symptoms; 3 of them stopped analgesics. In conclusion, the highest risk and grade of bortezomib neurotoxicity was seen in patients with baseline PN secondary to prior thalidomide use and DM. On the other hand, responding patients who received bortezomib in combination with thalidomide remained longer on therapy with minimal dose reductions. Although this may represent a section bias it is possible that thalidomide’s known anti-inflammatory and anti-angiogenesis properties protect against bortezomib-induced PN. Similar effects may explain the unexpected symptomatic improvement of PN on lenalidomide.

Bcl-2 acts as an important regulator of the mitochondrial pathway of apoptosis and promotes resistance of MM cells to chemotherapy. The Bcl-2 antisense oligonucleotide G3139 specifically targets Bcl-2 and may enhance the anti-tumor efficacy of Dex and Thal. In this trial G3139 was administered at 5 mg to the first 3 Pts and then 7 mg/kg/d by IVCI for 7d of 21d cycle. On day 4, Pts started Dex 40 mg daily for 4 d and Thal 100-400 mg as tolerated. After 3 cycles, responding Pts continued G3139 on a 5-week cycle with Dex 20 mg x 4d and Thal at the tolerated daily dose for up to 1 yr with an optional second yr for responding Pts. Thirty-three Pts treated to date had the following characteristics: median age 60 yrs (range: 28- 76), 22 males; 16 Pts had complex karyotypes; 14 Pts had B2M > 2.5 g/dl; LDH >1.5 normal in 7 Pts; Cr >1.5 mg/dl in 6 Pts; platelets grade 2) included fatigue, neutropenia, fever, electrolyte disturbance, muscle cramps, rash, hypotension, constipation and infections. Only 3 Pts maintained 400 mg/d of Thal, most Pts required dose reduction to 50–200 mg/d due neuropathy. Thirty Pts were evaluable for response; 24 Pts (80%) had documented responses, including 2 CR, 4 near-CR (+ immunofixation) and 12 PR; 6 had minimal response and 6 Pts had PD. The median duration of response is 13 mos. The estimated PFS is 12 mos and the median OS is 17.4 mo. The upper limits of the 95% confidence interval for PFS and OS have not been reached. At a median follow up of 1 yr (range 1.5–16.6 mo), 7 Pts had died and 26 are alive, of them 16 Pts continue on the study. Responding Pts had an early and significant increase in polyclonal IgM from a median baseline of 35.5 mg/dl (range: 8–75) to 94 (45–211) after cycle 3 (P=0.005), suggesting activation of the innate immune system. CD138+ cells were isolated from BM aspirates pre-treatment, and on d 4 or 7, and 28. Western blot analysis of Bcl-2 protein demonstrated demonstrated a decrease in Bcl-2 levels after normalization for protein loading by densitometry in 3 of 7 Pts with sufficient cells for analysis. The change of Bcl-2 protein levels did not correlate with response. Real time quantitative RT-PCR analysis was subsequently used to evaluate changes in Bcl-2 gene expression. Of 9 Pts evaluated, 6 demonstrated a significant decrease in Bcl-2 mRNA expression when normalized against GAPDH; 5 of them had a clinical response. In conclusion, G3139, Dex and Thal regimen is well tolerated in relapsed MM Pts. G3139 was associated with significant decrease in Bcl-2 gene expression in some Pts. G3139 appears to over-come resistance to Dex/Thal with impressive clinical responses in relapsed/refractory MM patients.

In vitro data suggest that the addition of bortezomib to chemotherapy can enhance the anti-myeloma activity achieved with either therapy alone. We hypothesized that combining bortezomib and DT-PACE in newly diagnosed MM can increase the CR rate and serve as a new mobilization regimen. The primary objective of the study is to determine the MTD of bortezomib (3 dose levels: 0.7, 1.0, 1.3 mg/m2 days 1, 4, and 8) in combination with thalidomide-based regimen (DT-PACE: dexamethasone 40 mg/day and thalidomide 200–400 mg/day orally x 4 days, Cisplatinum 10 mg/m2, Adriamycin 10 mg/m2, Cyclophosphamide 400 mg/m2 and Etoposide 40 mg/m2 all given by IVCI for 4 days). G-CSF 10 ug/kg/day was given from day 5 until stem cell collection or ANC > 2000, G-CSF was held on day 8 for Bortezomib dose. The secondary endpoint was to evaluate the effects of the mobilization regimen on the cellular composition of the graft and subsequent engraftment after high-dose chemotherapy. Five Pts have been enrolled on the study, median age 55 (range: 55–69), all had received 1–2 cycle of thalidomide based therapy, 2 had PD and 3 had PR. Three Pts were enrolled on dose level I; one Pt had grade III diarrhea and DVT with cycle one and tolerated cycle 2 well. One Pt had syncope with cycle 2. Two additional Pts were enrolled at dose level I with no grade III toxicity. After cycle 1, Pts underwent stem cell collections on day 13 (2 Pts had 1 collection and 3 had 2-day collection). Four Pts had a median of 21.7x 106 CD 34+/kg (range: 18.8–33.3) collected. One Pt continued thalidomide after day 4 through mobilization; he collected 4.3 x 106 CD 34+/kg. These collections compares favorably to those obtained from 14 Pts who collected stem cells after DT-PACE at our center in the past yr. This retrospective control group had a median of 14.5 days to collection (range: 11– 22d) with a median 17.8 x 106 CD 34+/kg (range: 9– 35). All 5 Pts responded to therapy; 3 had near CR and 2 PR after cycle 2. To date, 3 Pts received melphalan 200 mg/m2 followed by 4.3–5 x 106 CD 34+/kg and GCSF 5 mg/kg/day SC from day 5 until ANC > 1000 x 2 days. The first patient developed CMV antigenemia treated with ganciclovir. He received a boost of 5 x 106 CD 34/kg on day 21 and engrafted ANC > 1000 on day 25 and Plt > 20000 by day 49. The second patient reached ANC > 1000 on day 24 and plt > 20,000 by day 31. He developed autologous GVHD clinical grade II (skin and gut) upon engraftment, biopsy proven. He responded to steroids. The third patient whose cells were collected on thalidomide, engrafted ANC> 1000 by day 12 and Plt> 20000 by day 21, he has Plt < 30000 at day +35. The other 2 Pts await transplant. In comparison, control Pts mobilized with DT-PACE Pts (n=14) reached ANC > 1000 at a median of 12 days (range: 11-20) and plt > 20000 at 18 days (range: 13–25). Colony counts from DVT-PACE collections yielded a median CFU-C of 525 x 104/kg (range: 118–1000) compared to 540 (range: 153–1388) for DT-PACE. The effects of Bortezomib on the mobilized stem cells is unclear at this time, the delayed engraftment is concerning and will be better defined after additional Pts are treated on this protocol. Theoretically Bortezomib could affect stem cell adhesion molecules resulting in adequate stem cell collections and CFU-C in vitro that later affect stem cell homing and subsequent engraftment.

The Bcl-2 family member Mcl-1 is a critical survival factor for MM cells. However, the functions of Mcl-1 that distinguish it from other anti-apoptotic factors remain unclear. We used fluorescence polarization (FP) to measure the affinity of purified Mcl-1 for amphipathic peptides derived from the BH3-domains of Bad, Bid, Bim, Bik and PUMA, and compared these results with data obtained with Bcl-XL. Functional assays were then performed with mitochondria isolated from 8226 MM cells or clones engineered to overexpress Mcl-1 or Bcl-XL. In contrast to Bcl-XL, Mcl-1 binds to BadBH3 and BikBH3 with very low affinity (>1 uM), confirmed by its inability to bind full-length Bad or Bik proteins. Mcl-1 exhibited high affinity for BimBH3 (90 ± 27 nM), and intermediate affinities for Bid and PUMA. Bcl-XL had intermediate affinities for each of the BH3-peptides tested (175–400 nM). Mitochondria from parental 8226 cells were very sensitive to BidBH3 and BimBH3 as assayed by cytochrome c (cyt c) release; BadBH3 and BikBH3 did not directly induce cyt c release. Mitochondria from 8226-Mcl-1 (which overexpress Mcl-1 5-fold) were very resistant to BidBH3 (even at 500 uM), suggesting that sequestration of BH3-peptide by Mcl-1 was not the sole mechanism of apoptosis inhibition. Paradoxically, Mcl-1-overexpressing mitochondria were more sensitive to BimBH3 (despite the higher affinity of Mcl-1 for Bim), and neither BadBH3 nor BikBH3 could potentiate BidBH3- or BimBH3-induced cyt c release from these mitochondria. In contrast, 8226-Bcl-XL mitochondria were more sensitive to BidBH3, were similarly resistant to BimBH3, and were sensitized by BadBH3 and BikBH3. Protein crosslinking of Mcl-1-overexpressing mitochondria after culture with BidBH3 indicated that peptide sequestration could account for resistance at low peptide concentrations (5–30 uM), but not at higher doses (≥100 uM) where a Bid-induced Bak conformational change occurred as confirmed by exposure of a trypsin-sensitive site. Despite this, formation of Bak multimers was inhibited. (Bax was not detected in 8226 mitochondria). BimBH3 (30 uM) induced detectable cyt c release and Bak dimerization in Mcl-1-overexpressing mitochondria, but 500 uM BimBH3 failed to enhance either effect, and most of the cyt c remained in the mitochondrial pellet. Taken together, these data demonstrate that Mcl-1 and Bcl-XL are not equivalent in their anti-apoptotic functions at the OMM of MM cells. Bad and Bik do not bind Mcl-1, and cannot displace other BH3-only peptides from Mcl-1, suggesting that Mcl-1 has evolved to specifically bind apoptosis-inducing BH3-only proteins in a resistant complex. Mcl-1 confers exceptional protection against BidBH3, and experiments are underway to confirm this with tBid protein. We propose a model in which low levels of BH3-peptide can be sequestered by Mcl-1, while at higher concentrations Mcl-1 binding capacity becomes saturated, allowing peptides to bind Bak and induce a conformational change. However pore formation is inhibited, perhaps by direct binding of Mcl-1 to Bak. To induce pore formation, BH3-peptides must dissociate the Mcl-1/Bak complex: BimBH3, which has a high affinity for Mcl-1 can perform this function, while BidBH3 cannot. This model is consistent with our measured affinity of Mcl-1 for BakBH3 as compared with its affinities for BimBH3 or BidBH3. We suggest that Mcl-1 prevents full cyt c release by preventing activation of the permeability transition pore complex.