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Chronic lymphocytic leukemia (CLL) is effectively treated with targeted therapies including Bruton tyrosine kinase inhibitors and BCL2 antagonists. When these become ineffective, treatment options are limited. Positive transcription elongation factor complex (P-TEFb), a heterodimeric protein complex composed of cyclin dependent kinase 9 (CDK9) and cyclin T1, functions to regulate short half-life transcripts by phosphorylation of RNA Polymerase II (POLII). These transcripts are frequently dysregulated in hematologic malignancies; however, therapies targeting inhibition of P-TEFb have not yet achieved approval for cancer treatment. VIP152 kinome profiling revealed CDK9 as the main enzyme inhibited at 100 nM, with over a 10-fold increase in potency compared with other inhibitors currently in development for this target. VIP152 induced cell death in CLL cell lines and primary patient samples. Transcriptome analysis revealed inhibition of RNA degradation through the AU-Rich Element (ARE) dysregulation. Mechanistically, VIP152 inhibits the assembly of P-TEFb onto the transcription machinery and disturbs binding partners. Finally, immune competent mice engrafted with CLL-like cells of Eµ-MTCP1 over-expressing mice and treated with VIP152 demonstrated reduced disease burden and improvement in overall survival compared to vehicle-treated mice. These data suggest that VIP152 is a highly selective inhibitor of CDK9 that represents an attractive new therapy for CLL.

Successes with anti-CD20 antibodies in chronic lymphocytic leukemia (CLL) and enhanced activity of Fc-engineered vs unmodified antibody therapy suggest a potentially impactful role for natural killer (NK) cells and other innate immune cells in controlling this disease. Stimulated NK cells have shown promise as a cellular therapy, but their application has been constrained by limited expansion capacity and low cytotoxic activity against CLL cells. Here, we demonstrate that both healthy donor-derived and CLL patient-derived NK cells expand rapidly when stimulated with feeder cells expressing membrane-bound interleukin-21 (mbIL-21) and have potent cytotoxic activity against allogeneic or autologous CLL cells. Combination with anti-CD20 antibodies significantly enhances NK recognition and killing of CLL targets. As any CLL immune therapy would likely be given in combination, we assess commonly used treatments and demonstrate that ibrutinib has mixed suppressive and protective effects on expanded NK cells, whereas expanded NKs are highly resistant to venetoclax. We demonstrate efficacy in vivo in 2 xenograft mouse models of human CLL that support building upon a regimen of venetoclax and obinutuzumab with mbIL-21–expanded NK cells. Collectively, these data support development of mbIL-21–expanded NKs combined with the CD20 antibody obinutuzumab and venetoclax in the treatment of CLL.

Cytotoxic T lymphocyte antigen 4 (CTLA4) is a major immune checkpoint and target for cancer immunotherapy. Although originally discovered and primarily studied on T cells, its role on other cell types has also been recognized in recent years. Here we describe an unexpected interaction between ibrutinib (a targeted inhibitor of Bruton tyrosine kinase [BTK]) and CTLA4 expression on malignant chronic lymphocytic leukemia (CLL) cells. Although BTK itself does play a role in CTLA4 expression in CLL, we demonstrate that ibrutinib’s main suppressive effect on CTLA4 protein expression and trafficking occurs through non-BTK targets influenced by this drug. This suppression is not seen in T cells, indicating a different mechanism of CTLA4 regulation in CLL vs T cells. Appreciating this distinct mechanism and the beneficial non-BTK effects of ibrutinib may contribute to understanding the immune benefits of ibrutinib treatment and lead to therapeutic approaches to improve immune function in patients with CLL by suppressing CTLA4 expression.

Overactivation of immune responses is a hallmark of autoimmune disease pathogenesis. This includes the heightened production of inflammatory cytokines such as Tumor Necrosis Factor α (TNFα), and the secretion of autoantibodies such as isotypes of rheumatoid factor (RF) and anticitrullinated protein antibody (ACPA). Fcγ receptors (FcγR) expressed on the surface of myeloid cells bind Immunoglobulin G (IgG) immune complexes. Recognition of autoantigen-antibody complexes by FcγR induces an inflammatory phenotype that results in tissue damage and further escalation of the inflammatory response. Bromodomain and extra-terminal protein (BET) inhibition is associated with reduced immune responses, making the BET family a potential therapeutic target for autoimmune diseases such as rheumatoid arthritis (RA). In this paper, we examined the BET inhibitor PLX51107 and its effect on regulating FcγR expression and function in RA. PLX51107 significantly downregulated expression of FcγRIIa, FcγRIIb, FcγRIIIa, and the common γ-chain, FcϵR1-γ, in both healthy donor and RA patient monocytes. Consistent with this, PLX51107 treatment attenuated signaling events downstream of FcγR activation. This was accompanied by a significant decrease in phagocytosis and TNFα production. Finally, in a collagen-induced arthritis model, PLX51107-treatment reduced FcγR expression in vivo accompanied by a significant reduction in footpad swelling. These results suggest that BET inhibition is a novel therapeutic approach that requires further exploration as a treatment for patients with RA.

Figure S1. Serum cytokine analysis; Figure S2. Flow cytometric analysis of immune cell types.

Supplementary Figures 1-3

The bromodomain and extra-terminal domain inhibitor JQ1 has marked antitumor activity against several hematologic malignancies as well as solid tumor models. Here, we investigated its activity in vitro and in vivo against models of childhood rhabdomyosarcoma and Ewing sarcoma. In vitro, JQ1 (but not the inactive enantiomer JQ1R) inhibited cell proliferation and increased G1 fraction of cells, although there was no correlation between cell line sensitivity and suppression of c-MYC or MYCN. In vivo, xenografts showed significant inhibition of growth during the period of treatment, and rapid regrowth after treatment was stopped, activity typical of antiangiogenic agents. Furthermore, xenografts derived from cell lines intrinsically resistant or sensitive to JQ1 in vitro had similar sensitivity in vivo as xenografts. Further investigation showed that JQ1 reduced tumor vascularization. This was secondary to both drug-induced downregulation of tumor-derived growth factors and direct effects of JQ1 on vascular elements. JQ1 suppressed VEGF-stimulated vascularization of Matrigel plugs in mice, and in vitro suppressed differentiation, proliferation, and invasion of human umbilical cord vascular endothelial cells (HUVEC). In HUVECs, JQ1 partially suppressed c-MYC levels, but dramatically reduced AP-1 levels and activity through suppression of the AP-1–associated protein FOSL1. Our data suggest that the antitumor activity of JQ1 in these sarcoma models is largely a consequence of its antiangiogenic activity. Mol Cancer Ther; 15(5); 1018–28. ©2016 AACR.

The immunomodulatory drug lenalidomide has demonstrated efficacy in patients with chronic lymphocytic leukemia (CLL), despite a lack of direct cytotoxic effects in vitro. The mechanism of lenalidomide efficacy in vivo is thought to occur via a combination of enhanced immune activity and an alteration of tumor cell–microenvironment interactions. We demonstrate in whole blood from patients with CLL that lenalidomide significantly depletes malignant B cells. Lenalidomide also induced production of interleukin-21 (IL21) and its mRNA in T cells from patients with CLL. In addition, lenalidomide enhanced upregulation of functional IL21 receptor (IL21R) on the cell surface and increased receptor mRNA in vitro. The in vitro combination of IL21 and lenalidomide enhanced IL21-mediated cytotoxicity toward CLL cells through a variety of mechanisms. We show association of cell death with upregulation of Bid by IL21, enhanced upregulation of Bid by the combination therapy, and diminished Lck and downstream BCR signaling activation of Syk and PLCG2. Collectively, we demonstrated an immune cell–tumor cell interaction through lenalidomide-mediated induction of IL21 and IL21R, with enhanced IL21-mediated cytotoxicity, which provides justification for this combination in clinical trials for patients with CLL. Cancer Immunol Res; 4(8); 698–707. ©2016 AACR.

Although the role of dopamine (DA) in malignant tumors has been reported, its function in premalignant lesions is unknown. Herein we report that the stimulation of DA D2 receptors in endothelial cells in ultraviolet B (UVB)-induced cutaneous lesions in mice significantly reduced the tumor number, tumor burden, and malignant squamous cell carcinoma in these animals. DA D2 receptor agonist inhibited VEGFA-dependent proangiogenic genes in vitro and in vivo. However, the mice pretreated with selective DA D2 receptor antagonist inhibited the actions of the agonist, thereby suggesting that the action of DA was through its D2 receptors in the endothelial cells. To our knowledge, this study is the first to report DA-mediated regulation of pathogenesis and progression of UVB-induced premalignant skin lesions.Prevention Relevance:This investigation demonstrates the role of dopamine and its D2 receptors in UVB induced premalignant squamous cell skin lesions and how DA through its D2 receptors inhibits the development and progression of these lesions and subsequently prevents squamous cell carcinoma of the skin.

This includes additional material for the paper including 1) Table 1. Microarray results for genes of interest; 2) Figure s1 showing decrease in IKZF1 and IKZF3 by lenalidomide; and Figure S2. Lenalidomide, IL-21, and the combination of lenalidomide + IL-21 induce different transcriptional signatures

Legends for Supplementary figures S1 and S2

mAbs can induce antibody-dependent cellular cytotoxicity (ADCC) via the innate immune system's ability to recognize mAb-coated cancer cells and activate immune effector cells. Lenalidomide is an immunomodulatory agent with the capacity to stimulate immune cell cytokine production and ADCC activity. This phase I trial evaluated the combination of cetuximab with lenalidomide for the treatment of advanced colorectal and head and neck squamous cell cancers (HNSCC). This trial included patients with advanced colorectal cancer or HNSCC. Treatment consisted of cetuximab 500 mg/m2 i.v. every two weeks with lenalidomide given orally days 1–21 on a 28-day cycle. Three dose levels of lenalidomide were evaluated (15, 20, 25 mg). Correlative studies included measurement of ADCC, FcγRIIIA polymorphism genotyping, measurement of serum cytokine levels, and flow cytometric analysis of immune cell subtypes. Twenty-two patients were enrolled (19 colorectal cancer, 3 HNSCC). Fatigue was the only dose-limiting toxicity. One partial response was observed and 8 patients had stable disease at least 12 weeks. The recommended phase II dose is cetuximab 500 mg/m2 with lenalidomide 25 mg daily, days 1–21. Correlative studies demonstrated a dose-dependent increase in natural killer cytotoxic activity with increasing doses of lenalidomide. Cetuximab and lenalidomide were well tolerated. There was a lenalidomide dose-dependent increase in ADCC with higher activity in patients enrolled in cohort 3 than those enrolled in cohorts 1/2. Although response was not a primary endpoint, there was evidence of antitumor activity for the combination therapy. Further investigation of lenalidomide as an immunomodulator in solid tumors is warranted. Mol Cancer Ther; 15(9); 2244–50. ©2016 AACR.

Supplementary Fig. S1. Therapeutic efficacy of RV and HDACi against cancer B cells; Supplementary Fig. S2. HDACi increase JAM-1 expression without affecting cell viability; Supplementary Fig. S3. RV+HDACi display synergistic anti-myeloma activity; Supplementary Fig. S4. Combination treatment of MCL cells with RV and HDAC inhibitors results in increased cell death, compared to single agent treatments.

Supplemental Figure Legends; Supplemental Figure 1. JQ1 induces G1 accumulation without enhancing the sub-G1 population in sensitive sarcoma cell lines. Cells were exposed for 24 Hr to 500 nM JQ1; Supplemental Figure 2. Quantitation of immunoblots from Figure 1B, and 1C. MYC proteins are normalized against GAPDH; Supplemental Figure 3. JQ1 inhibits angiogenesis in rhabdomyosarcoma xenografts. CD34-positive staining (left panels; magnification 417X) and quantitation of IHC (right panels) for Rh10, and Rh28 rhabdomyosarcoma xenografts after 7 or 14 daily treatments with JQ1 or no treatment (controls); Supplemental Figure 4. JQ1 inhibits angiogenesis in Ewing sarcoma xenografts. CD34- positive staining (left panels; magnification 417X) and quantitation of IHC (right panels) for EW-5 and EW-8 Ewing sarcoma xenografts after 7 or 14 daily treatments with JQ1 or no treatment (controls); Supplemental Figure 5. Summary of JQ1 induced changes in angiogenic factors as determined by angiogenesis arrays in xenografts harvested from mice after 14 days treatment with JQ1, or HUVECs in vitro (0.5 μM 24 hr); Supplemental Figure 6. JQ1 does not inhibit the proliferative compartment in rhabdomyosarcoma xenografts. Ki67-positive staining (left panels; magnification 417X) was quantitated for Rh10, and Rh28 xenografts (right panels; magnification 417X) after 7 or 14 daily treatments with JQ1 or no treatment (controls); Supplemental Figure 7. JQ1 does not inhibit the proliferative compartment in Ewing sarcoma xenografts. Ki67-positive staining (left panels; magnification 417X) was quantitated for EW-5 and EW-8 xenografts (right panels; magnification 417X) after 7 or 14 daily treatments with JQ1 or no treatment (controls).

Supplementary file

Supplemental Figure 1 PDF file 151K, Photomicrograph. IGF-2 circumvents CP01-B02 antibody in vivo

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Supplemental Table 1 PDF file 56K, Comparisons of time-to-event curves for single or combined antibody therapy against pediatric sarcoma xenografts (listed are adjusted p-values)

PDF file - 9027K, Supplemental Table 1 and supplemental Figures 1-3 Supplemental Table 1. MEK functional activation gene signature qRT-PCR results Supplemental Figure 1. For individual growth curve plots: Upper left panel: Control; Upper right panel: AZD6244 100 mg/kg BID x 5/SID x 2 for six consecutive weeks; lower left panel: AZD6244 75 mg/kg BID x 5/SID x 2 for six consecutive weeks; Lower right panel100 mg/kg SID for six consecutive weeks. Tumors A5 and B4 (arrowed) were selected for further transplant to select for selumetinib resistance in vivo. Supplemental Figure 2. Representative immunostaining of BT-40 and derivative xenografts using the VE1 antibody that recognizes BRAFV600E.Tumor samples were fixed and stained as described in a Materials and Methods and read 'blinded' (DC). Panels at the bottom were stained simultaneously and demonstrate intensity of staining for patient derived brain tumors. Supplemental Figure 3. qRT-�PCR to detect IL6 receptor alpha was performed using human-specific primers on snap-�frozen control tumor tissue as described in Supplemental Table 1.

Supplemental Figure 5 PDF file 132K, Photomicrograph

Supplemental Figure 3 PDF file 121K, Photomicrograph

Supplemental Figure S1. Levels of serum cytokines in patients over the course of treatment. Serum levels of the cytokines IFN-γ (A), IP-10 (B), TNF-α (C) MIP-1α (D), MIP-1β (E), RANTES (F), GM CSF (G), and IL-8 (H) were measured at baseline and during cycle 8 using a custom V-Plex assay. Assay was performed in triplicate and the average was plotted for each individual patient. *, p=0.0008 for IFN-γ, p=0.0011 for IP-10, p=0.0102 for TNF-α Supplemental Figure S2. Levels of serum cytokines in patients stratified by length of progression-free survival. Serum levels of the cytokines MIP-1α (A), MIP-1β (B), RANTES (C), GM-CSF (D), and IL-8 (E) were measured at baseline and during cycle 8 using a custom V-Plex assay. The assay was performed in triplicate and the average was plotted for each individual patient stratified by progression-free survival greater than or less than 100 days Supplemental Figure S3. Gating strategy for defining monocytic and granulocytic MDSC. Examples of monocytic-dominant (A) and granulocytic-dominant (B) total MDSC. PBMCs procured from pre-therapy blood draws were stained with anti-CD33-APC, anti-HLA-DR-PECy7, anti-CD11b-PE, anti-CD14-V-450, and anti CD15-FITC antibodies; CD33+/HLA-DR- populations were further characterized by CD15, CD14, and CD11b expression. Percentages gleaned from CD15+CD11b+ and CD14+CD11b+ quadrants were back multiplied by total MDSC (CD33+HLA-DR-) to obtain values presented in Table S3.

Supplementary Figure 1. SMARCA4 (212520_s_at) expression correlates with overall survival independent of stage in the Director's Challenge Study. Supplementary Figure 2. Low SMARCA4 (214360_at) expression correlates with decreased overall survival in the Director's Challenge Study. Supplementary Figure 3. Low SMARCA4 (208794_s_at) expression correlates with improved disease-specific survival with adjuvant cisplatin-based chemotherapy in the JBR.10 trial. Supplementary Figure 4. Low SMARCA4 (208794_s_at) expression correlates with improved overall survival with adjuvant cisplatin-based chemotherapy in the JBR.10 trial. Supplementary Figure 5. Low SMARCA4 (208793_x_at (A), 212520_s_at (B), 214360_at (C), and 215714_s_at (D)) expression correlates with improved disease-specific survival with adjuvant cisplatin-based chemotherapy in the JBR.10 trial. Supplementary Figure 6. SMARCA2 (206543_at) expression trends toward improved disease-specific survival with adjuvant cisplatin-based chemotherapy in the JBR.10 trial. Supplementary Figure 7. Mapping of the probe sets to the SMARCA4 gene.

Figure Legends for Supplementary data

Background: In pediatric tumor xenograft models, tumor-derived insulin growth factor (IGF-2) results in intrinsic resistance to IGF-IR–targeted antibodies, maintaining continued tumor angiogenesis. We evaluated the antiangiogenic activity of a ligand-binding antibody (MEDI-573) alone or in combination with IGF-I receptor binding antibodies (MAB391, CP01-B02).Methods: IGF-stimulated signaling was monitored by increased Akt phosphorylation in sarcoma and human umbilical cord vascular endothelial cells (HUVEC). Angiogenesis was determined in vitro using capillary tube formation in HUVECs and in vivo using a VEGF-stimulated Matrigel assay. Tumor growth delay was examined in 4 sarcoma xenograft models.Results: The IGF ligand-binding antibody MEDI-573 suppressed Akt phosphorylation induced by exogenous IGF-I and IGF-2 in sarcoma cells. Receptor-binding antibodies suppressed IGF-I stimulation of Akt phosphorylation, but IGF-2 circumvented this effect and maintained HUVEC tube formation. MEDI-573 inhibited HUVEC proliferation and tube formation in vitro, but did not inhibit angiogenesis in vivo, probably because MEDI-573 binds murine IGF-I with low affinity. However, in vitro antiangiogenic activity of MEDI-573 was also circumvented by human recombinant IGF-I. The combination of receptor- and ligand-binding antibodies completely suppressed VEGF-stimulated proliferation of HUVECs in the presence of IGF-I and IGF-2, prevented ligand-induced phosphorylation of IGF-IR/IR receptors, and suppressed VEGF/IGF-2–driven angiogenesis in vivo. The combination of CP1-BO2 plus MEDI-573 was significantly superior to therapy with either antibody alone against IGF-I and IGF-2 secreting pediatric sarcoma xenograft models.Conclusions: These results suggest that combination of antibodies targeting IGF receptor and ligands may be an effective therapeutic strategy to block angiogenesis for IGF-driven tumors. Clin Cancer Res; 19(11); 2984–94. ©2013 AACR.

Supplemental Figure 2 PDF file 143K, Western Blot

PDF file - 1614K, Methylated CpG sites in the Nestin promoter region of rQNestin34.5, immediately upstream of the gamma1 34.5 gene.

PDF file - 7683K, HSV Antigen immunohistochemistry.

Purpose:Infectious complications constitute a leading cause of morbidity and mortality in chronic lymphocytic leukemia (CLL). Patients respond poorly to vaccines, particularly pneumococcal polysaccharide and influenza vaccines. In addition, patients with genetically high-risk disease are at increased risk for early disease progression and death. Lenalidomide, an oral immunomodulatory agent with demonstrated clinical activity in CLL, can potentially restore immune system dysfunction associated with CLL while improving disease outcomes.Patients and Methods:Phase II study randomized 49 patients with genetically high-risk CLL or small lymphocytic lymphoma [SLL; defined as unmutated Ig heavy chain variable region, deletion(17p) or (11q), and/or complex abnormal karyotype], to receive lenalidomide either concurrent (arm A) or sequential to (arm B) two doses of 13-valent protein-conjugated pneumococcal vaccine (PCV13) administered 2 months apart, in patients not meeting International Workshop on Chronic Lymphocytic Leukemia treatment criteria.Results:Four serotypes (3, 4, 5, 6B) achieved the additional seroprotection definition of a fourfold increase in arm A, and six serotypes (3, 4, 5, 6B, 19A, 19F) in arm B. All patients achieved the defined concentration of 0.35 μg/mL for at least one serotype tested. No significant difference was observed with the addition of lenalidomide. At median time on treatment of 3.6 years, median progression-free survival (PFS) was 5.8 years [95% confidence interval (CI), 3.1—not reached]. PFS at 1, 2, and 3 years was 85% (95% CI, 72–93), 79% (95% CI, 64–88), and 72% (95% CI, 57–83), respectively.Conclusions:Lenalidomide is efficacious with manageable toxicities as an early intervention strategy in patients with high-risk CLL, but did not enhance humoral response to PCV13 vaccine.

Supplemental Figure 4 PDF file 103K,Histogram

Purpose: The BT-40 low-grade childhood astrocytoma xenograft model expresses mutated BRAFV600E and is highly sensitive to the MEK inhibitor selumetinib (AZD6244). In this study, we developed and characterized selumetinib resistance and explored approaches to circumventing the mechanisms of acquired resistance.Experimental Design: BT-40 xenografts were selected in vivo for selumetinib resistance. Resistant tumors were obtained and characterized, as were tumors that reverted to sensitivity. Characterization included expression profiling, assessment of MEK signature and compensatory pathways, MEK inhibition, BRAF expression, and cytokine levels. Combination treatment of BT-40/AZD–resistant tumors with the MEK inhibitor and a STAT3 inhibitor (LLL12) was assessed.Results: Resistance was unstable, tumors reverting to selumetinib sensitivity when passaged in untreated mice, and MEK was equally inhibited in sensitive and resistant tumors by selumetinib. Drug resistance was associated with an enhanced MEK signature and increased interleukin (IL)-6 and IL-8 expression. Selumetinib treatment induced phosphorylation of STAT3 (Y705) only in resistant xenografts, and similar results were observed in BRAFV600E astrocytic cell lines intrinsically resistant to selumetinib. Treatment of BT-40–resistant tumors with selumetinib or LLL12 had no significant effect, whereas combined treatment induced complete regressions of BT-40/AZD–resistant xenografts.Conclusions: Resistance to selumetinib selected in vivo in BT-40 tumor xenografts was unstable. In resistant tumors, selumetinib activated STAT3, and combined treatment with selumetinib and LLL12 induced complete responses in resistant BT-40 tumors. These results suggest dual targeting BRAF (V600E) signaling and STAT3 signaling may be effective in selumetinib-resistant tumors or may retard or prevent onset of resistance. Clin Cancer Res; 19(24); 6716–29. ©2013 AACR.

Supplemental Figure Legends