Prolonged early G1 arrest by selective CDK4/CDK6 inhibition sensitizes myeloma cells to cytotoxic killing through cell cycle–coupled loss of IRF4 by Xiangao Huang, Maurizio Di Liberto, David Jayabalan, Jun Liang, Scott Ely, Jamieson Bretz, Arthur L. Shaffer, Tracey Louie, Isan Chen, Sophia Randolph, William C. Hahn, Louis M. Staudt, Ruben Niesvizky, Malcolm A. S. Moore, and Selina Chen-Kiang Blood Volume 120(5):1095-1106 August 2, 2012 ©2012 by American Society of Hematology

Inhibition of CDK4/CDK6 induces early G1 arrest and cell-cycle synchronization. Inhibition of CDK4/CDK6 induces early G1 arrest and cell-cycle synchronization. (A) Schema for reversible inhibition of CDK4/CDK6 by PD 0332991 (PD). D indicates cyclin D. (B) MM1.S cells were cultured with PD for 24 hours (h) and released into fresh media for the time indicated. *BrdU was added 30 minutes before cell harvest for FACS analysis of BrdU uptake and DNA content per cell. Number in the FACS profile indicates the percentage of BrdU-positive cells. (C) Immunoblotting and (D) quantitative RT-PCR analysis of cells cultured as in panel B. pSRb indicates Rb phosphorylated on serine 807/811. (E) Hierarchical clustering analysis of gene expression in MM1.S cells cultured with PD for 12 hours (12hPD) or 24 hours (24hPD), or released into fresh medium for 4 hours (4hpo24hPD) or 18 hours (18hpo24hPD) after 24 hours of PD treatment. The 2797 genes included in this analysis have a log2(treated/control) value > 1 in at least 1 of 3 independent arrays and a P value < .05. (F) Scatter plot of Log2 (12hPD/Cntl) and Log2(24hPD/Cntl) after normalization to the expression of ACTB (β-actin). The middle line indicates no variation, and the outer 2 lines indicate a 2-fold variation between the log ratio values. (G) Heat maps of genes from panel E. Data are representative of 6 independent experiments. Xiangao Huang et al. Blood 2012;120:1095-1106 ©2012 by American Society of Hematology

Sensitization of myeloma cells to cytotoxic killing in pG1 and pG1-S. Sensitization of myeloma cells to cytotoxic killing in pG1 and pG1-S. (A) MM1.S cells prolonged arrest in early G1 (pG1) by PD (24 hours), synchronized into S-phase for the time indicated (pG1-S), or without PD treatment (Cntl), were pulsed with bortezomib (BTZ, 60nM) for 1 hour or left untreated (No BTZ). Cell death was determined by the loss of MitoTracker Red (MT−) at 7 hours from BTZ pulsing. (B) “% Live cells” indicates the number of live cells relative to untreated Cntl cells determined after BTZ pulsing. (C) HMCL CAG and KMS12PE cells were pulsed with BTZ (120nM) for 1 hour in pG1-S (4 hours after PD withdrawal), and cell death was determined by ToPro-3 staining at 18 hours after BTZ pulsing. The 2132 cells were cultured similarly except that BTZ was used at 350nM and cell death was determined by annexin V staining. (D) FACS analysis of BrdU uptake and DNA content in pG1 and pG1-S MM1.S cells cultured as indicated in the schema. (E) MM1.S cells were pulsed with BTZ (1 hour, 60 or 120nM) at 4 hours after release from PD pretreatment for the time indicated. The percentages of live and dead cells were determined as in panel B, using cells untreated by PD or BTZ as a reference. (F) MM1.S cells were cultured with low BTZ with or without PD pretreatment as diagrammed, and analyzed for live cells as in panel E and BrdU uptake as in panel D. (G) RPMI8226 cells were cultured with 0.5μM PD for 24 hours and released into fresh media for 12 hours. The cells were then treated with BTZ (20nM) for 8 hours, and the percentage of MT− cells was determined. Data are representative of 5 independent experiments. Xiangao Huang et al. Blood 2012;120:1095-1106 ©2012 by American Society of Hematology

Inhibition of CDK4/CDK6 is the basis for sensitization to cytotoxic killing. Inhibition of CDK4/CDK6 is the basis for sensitization to cytotoxic killing. MM1.S cells were infected with CDK4, CDK6, or a nontargeting (n-t) shRNA (sh) lentivirus. (A) Immunoblotting and BrdU+ uptake at 66 hours after infection. (B) Percentage of live cells was determined at 17 hours of BTZ (4nM) treatment starting at 72 hours after infection, using the n-t shRNA lentivirus-infected cells without treatment as a control. (C) Left: Rb protein expression in MM1.S and U266 cells. Middle: Percentage of S phase and viable U266 cells after culturing with PD for 24 hours relative to input. Right: MT− U266 cells after BTZ treatment (24 hours) in the absence (Cntl) or presence of PD pretreatment (pG1; 0.5μM, 24 hours). (D) Caspase and poly ADP-ribose polymerase cleavage in MM1.S cells after BTZ treatment (12 hours) in pG1 (PD, 24 hours) or left untreated (Cntl), in the presence or absence of HS-5 BMSCs. (E) Q-VD-OPh (20μM) or DMSO was added to MM1.S cells in pG1 or left untreated for 1 hour before BTZ pulsing (120nM). MT− cells were determined at 7 hours from BTZ pulsing. Data are representative of 3 independent experiments. Xiangao Huang et al. Blood 2012;120:1095-1106 ©2012 by American Society of Hematology

Bim enhances bortezomib-induced apoptosis in the absence of Noxa in pG1 and in cooperation with Noxa in pG1-S. Bim enhances bortezomib-induced apoptosis in the absence of Noxa in pG1 and in cooperation with Noxa in pG1-S. (A) Quantitative RT-PCR analysis of PMAIP1 and BCL2L11 mRNAs in MM1.S cells cultured as in Figure 1B. (B-C) Quantitative RT-PCR analysis of Bcl-2 family genes and immunoblotting of Bcl-2 family proteins and cleaved caspase-3 at 7 hours from BTZ pulsing (60nM) in control, pG1 or pG1-S (12 hours after PD withdrawal) cells. (D) Noxa mRNA and protein expression in MM1.S cells at 72 hours after infection with PMAIP1 or GFP shRNA lentivirus. (E) PMAIP1 or GFP shRNA (sh) lentivirus-infected MM1.S cells were treated with PD and BTZ (60nM) as indicated. MT− and live cells were determined at 7 hours from BTZ pulsing. (F-G) BCL2L11 or GFP shRNA (sh) lentivirus-infected MM1.S cells were treated with PD and BTZ (60nM) as indicated. Bim protein levels and the percentages of MT− cells were determined as indicated. (H) Immunoblotting of Bim in MM1.S and LP-1 myeloma cells (left). LP-1 cells were pulsed with BTZ (120nM) in pG1 (0.5μM PD, 24 hours) or pG1-S (4 hours after PD withdrawal). The percentage of live cells relative to non-BTZ–treated cells was determined 18 hours from BTZ pulsing (right). Data are representative of 3 independent experiments. Xiangao Huang et al. Blood 2012;120:1095-1106 ©2012 by American Society of Hematology

Cooperative regulation of IRF4 by the cell cycle and bortezomib. Cooperative regulation of IRF4 by the cell cycle and bortezomib. Quantitative RT-PCR analysis and immunoblotting of IRF4 expression in MM1.S cells (A) cultured as in Figure 1B, and (B) at 7 hours from pulsing with BTZ (1 hour, 120nM) in pG1 (PD, 24 hours) or in pG1-S (4 hours after PD withdrawal). (C) IRF4 protein level, MT− and live cells were determined in MM1.S cells at 72 hours after infection with IRF4 or the control GFP shRNA lentivirus. (D) MM1.S cells were treated with PD and pulsed with BTZ (60nM) after lentivirus infection as indicated. Percentage of live cells was determined using cells infected with the GFP shRNA lentivirus as a control. (E) KMS12PE cells infected with a retrovirus expressing human IRF4 (ToIRF4) or the control virus (Vxy) were treated with doxycycline (Dox, 20 ng/mL), PD (0.3μM), or BTZ (250nM) as indicated. The IRF4 protein level was determined by immunoblotting and cell death by ToPro-3 staining. Data are representative of 3 independent experiments. Xiangao Huang et al. Blood 2012;120:1095-1106 ©2012 by American Society of Hematology

CDK4/CDK6 inhibition accelerates G1 arrest and enhances bortezomib killing of primary myeloma cells. CDK4/CDK6 inhibition accelerates G1 arrest and enhances bortezomib killing of primary myeloma cells. Primary CD138+ BM myeloma cells isolated from individual patients (MM, referred to by the number at the bottom of each graph) were pretreated with 0.5μM PD for 4 or 24 hours in the HS-5 BMSC coculture before addition of BTZ and cultured for an additional 24 hours unless otherwise indicated. (A) BrdU uptake in MM cells treated with PD for 16 hours, with BrdU presented in the last 13 hours. (B-D) Viability of MM cells after BTZ addition. (E) Viability of MM cells after culturing as indicated (left); immediately after isolation (−24), at 24 hours after incubation with PD (0), and at 24 and 48 hours after further incubation with BTZ (4nM) and PD (middle), and ToPro-3 analysis at 48 hours of BTZ treatment (right). (F) Viability after BTZ addition in MM cells pretreated with PD for time indicated. (G) Immunoblotting of IRF4 at 12 hours after BTZ addition in MM10 (as shown in panel F) with or without 24-hour PD pretreatment. Numbers indicate the relative level of IRF4 compared with cells left untreated by PD or BTZ and corrected for loading by the actin signal. Data represent mean ± SD in triplicate. P value was determined by 2-tailed or 1-tailed (*) t test. Xiangao Huang et al. Blood 2012;120:1095-1106 ©2012 by American Society of Hematology

Induction of pG1 and pG1-S enhances tumor suppression by bortezomib. Induction of pG1 and pG1-S enhances tumor suppression by bortezomib. (A) A schema for treatment of NOD/SCID mice developing aggressive tumor after injection with Luc+GFP+MM1.S cells with PD (150 mg/kg) and BTZ (0.25 mg/kg), and the time (day) of BLI and MT− (FACS) analyses. (B) IHC of IRF4 (red) and phospho-Rb (Ser807/811; pSRb, blue) or Ki67 (blue) in MM1.S cells from BM of mice treated as indicated. The percentages of pSRb+ or Ki67+ cells in IRF4+ myeloma cells are indicated. (C) Fold of tumor growth represents BLI on day 9 relative to that on day 1 of the same mice. (D) BM GFP+ MM1.S cells and BM cells were flushed from femurs on day 11, stained with MitoTracker Red, and analyzed by FACS. Number indicates the percentage of MT− cells (mean ± SD). (E) Schema for treatment of myeloma-developing NOD/SCID mice with PD (80 mg/kg) and BTZ (0.25 mg/kg). (F) BLI of tumors in mice on days 1 and 22. (G) Bioluminescence representing tumor mass (photons/s/cm2/steradian) on days indicated. V indicates ventral; and D, dorsal. P value was determined by 2-tailed or 1-tailed (*) t test. Data are representative of 3 independent experiments. Xiangao Huang et al. Blood 2012;120:1095-1106 ©2012 by American Society of Hematology