Zhi Hua Li

Preclinical studies of fibroblast growth factor receptor 3 as a therapeutic target in multiple myeloma

Dysregulation of fibroblast growth factor receptor 3 (FGFR3) by the translocation t(4;14)(p16;q32) occurs in 15% of multiple myeloma (MM) patients and confers a growth and survival advantage to malignant plasma cells. As FGFR3 is a molecular target, we assessed the therapeutic potential of the FGFR‐specific tyrosine kinase inhibitors SU5402 and SU10991 in MM. SU5402 inhibited FGFR3 phosphorylation in vitro and in murine MM tumour models. B cells dependent on FGFR3 for survival were specifically sensitive to SU5402. A panel of 11 human myeloma cell lines was studied, five bearing the t(4;14) translocation. The KMS11 human myeloma cell line, which expresses constitutively active mutant FGFR3, displayed an 85% decrease in S‐phase cells, a 95% increase in G0/G1 cells, and 4·5‐fold increase in apoptotic cells after 72 h treatment with 10 μmol/l SU5402. Activated extracellular signal‐regulated kinases 1 and 2 and signal transducer and activator of transcription 3 were rapidly down‐regulated after SU5402 treatment. In human myeloma cell lines expressing wild‐type FGFR3 the stimulating effect of aFGF ligand was abrogated by SU5402 treatment. Myeloma cells lacking the t(4;14) or with the t(4;14) and a secondary RAS mutation did not respond to therapy. These findings support the development of clinical trials of early intervention with FGFR3 inhibitors in t(4;14) myeloma.

A phase I trial of adenovector-mediated delivery of interleukin-2 (AdIL-2) in high-risk localized prostate cancer

Preclinical studies demonstrate that intratumoral delivery of adenovirus expressing IL-2 eradicates pre-established tumors in mice and confers immune protection from rechallenge. To explore the activity of AdCAIL-2 in prostate cancer, a Phase I clinical trial was conducted in patients with localized disease and Gleason score >7 or prostate-specific antigen (PSA) >10 plus Gleason score 7. A total of 12 patients were injected 4 weeks prior to prostatectomy in a dose-escalation study at doses of 109, 5 × 109 and 1010 PFU of virus. No dose-limiting toxicity was observed. Side effects included perineal discomfort, hematuria, flu-like symptoms in two patients and urinary hesitancy in one patient. Pathology demonstrated an inflammatory response consisting predominantly of CD3+CD8+ T lymphocytes with areas of tumor necrosis. Intracellular cytokine staining of tumor-infiltrating lymphocytes demonstrated increases in both γ-interferon and IL-4 secreting T cells after vaccination. PSA levels fell in five of five evaluable patients treated at the lowest dose (mean decline of 33.3%, range 17–69%). At higher doses, PSA values initially increased after injection, then fell to baseline prior to surgery. This trial demonstrates the feasibility and safety of intraprostatic adenovector-mediated IL-2 gene delivery.

Identification of a non-phosphorylated, cell permeable, small molecule ligand for the Stat3 SH2 domain

Signal transducer and activator of transcription 3 (Stat3) protein is a cytosolic transcription factor that is aberrantly activated in numerous human cancers. Inhibitors of activated Stat3-Stat3 protein complexes have been shown to hold therapeutic promise for the treatment of human cancers harboring activated Stat3. Herein, we report the design and synthesis of a focused library of salicylic acid containing Stat3 SH2 domain binders. The most potent inhibitor, 17o, effectively disrupted Stat3-phosphopeptide complexes (K(i)=13 μM), inhibited Stat3-Stat3 protein interactions (IC(50)=19 μM) and silenced intracellular Stat3 phosphorylation and Stat3-target gene expression profiles. Inhibition of Stat3 function in both breast and multiple myeloma (MM) tumor cells correlated with induced cell death (EC(50)=10 and 16 μM, respectively).

The Tricyclic Antidepressant Amitriptyline Inhibits d-Cyclin Transactivation and Induces Myeloma Cell Apoptosis by Inhibiting Histone Deacetylases: In Vitro and In Silico Evidence

Amitriptyline is a classic tricyclic antidepressant (TCA) and has been used to treat the depression and anxiety of patients with cancer, but its relevance to cancer cell apoptosis is not known. In the present study, we demonstrated that amitriptyline inhibited cyclin D2 transactivation and displayed potential antimyeloma activity by inhibiting histone deacetylases (HDACs). Amitriptyline markedly decreased cyclin D2 promoter-driven luciferase activity, reduced cyclin D2 expression, and arrested cells at the G0/G1 phase of the cell cycle. Amitriptyline-induced apoptosis was confirmed by Annexin V staining, and cleavage of caspase-3 and poly(ADP-ribose) polymerase-1. d-Cyclin expression is reported to be epigenetically regulated by histone acetylation. Thus, we examined the effects of amitriptyline on histone 3 (H3) acetylation and demonstrated that amitriptyline increased acetylation of H3 and expression of p27 and p21. Further studies indicated that amitriptyline interfered with HDAC function by down-regulation of HDAC3, -6, -7, and -8, but not HDAC2, and by interacting with HDAC7. Molecular docking analysis and molecular dynamics simulations revealed that amitriptyline bound to HDAC7 and formed strong van der Waals interactions with five residues of HDAC7, including Phe162, His192, Phe221, Leu293, and His326, thus inhibiting HDAC activity. Therefore, we found that amitriptyline inhibited cyclin D2 transactivation and HDAC activity and could be a promising treatment for multiple myeloma.

Tandem immunoprecipitation of phosphotyrosine-mass spectrometry (TIPY-MS) indicates C19ORF19 becomes tyrosine-phosphorylated and associated with activated epidermal growth factor receptor.

To identify phosphotyrosine (pY) sites in the epidermal growth factor receptor (EGFR) network, a tandem immunoprecipitation-mass spectrometry method (TIPY-MS) was applied wherein protease-digested EGFR immune complexes were extracted with anti-pY after Rush et al. ( Nat. Biotech. 2005, 23, 94 ) and analyzed by LC-MS/MS. New pY sites in the pathway were found, including SOS1 Y1065, SOS2 Y1275, CBL-B Y889, and in the EGFR regulatory protein Mig-6 Y458. The novel human C19orf19 gene product was found EGFR-associated and phosphorylated at 5 tyrosines in response to EGFR activation and, therefore, represents a new component of the EGFR signaling network.

Circulating tumour DNA sequence analysis as an alternative to multiple myeloma bone marrow aspirates

The requirement for bone-marrow aspirates for genomic profiling of multiple myeloma poses an obstacle to enrolment and retention of patients in clinical trials. We evaluated whether circulating cell-free DNA (cfDNA) analysis is comparable to molecular profiling of myeloma using bone-marrow tumour cells. We report here a hybrid-capture-based Liquid Biopsy Sequencing (LB-Seq) method used to sequence all protein-coding exons of KRAS, NRAS, BRAF, EGFR and PIK3CA in 64 cfDNA specimens from 53 myeloma patients to >20,000 × median coverage. This method includes a variant filtering algorithm that enables detection of tumour-derived fragments present in cfDNA at allele frequencies as low as 0.25% (median 3.2%, range 0.25–46%). Using LB-Seq analysis of 48 cfDNA specimens with matched bone-marrow data, we detect 49/51 likely somatic mutations, with subclonal hierarchies reflecting tumour profiling (96% concordance), and four additional mutations likely missed by bone-marrow testing (>98% specificity). Overall, LB-Seq is a high fidelity adjunct to genetic profiling of bone-marrow in multiple myeloma.

RU486-inducible retrovirus-mediated caspase-3 overexpression is cytotoxic to bcl-xL-expressing myeloma cells in vitro and in vivo.

The antiapoptotic protein bcl-x(L) is upregulated in a variety of solid tumors and in primary hematologic malignancies such as multiple myeloma. Activated caspase-3 cleaves proteins essential for cell survival, including bcl-x(L). To explore the potential of caspase-3 as a cytotoxic and immunostimulatory molecule in the treatment of malignancy, an RU486-inducible caspase-3 retrovirus was constructed, validated, and used to transduce first 3T3 and subsequently murine myeloma B9BM1 cells (creating the cell line B9BM-C3). After induction, apoptotic cell death of 3T3 and B9BM-C3 cells began by 4 h and was complete by 48 h postinduction, while nontransduced cells remained viable. Annexin V staining demonstrated 43, 76, and 98% apoptotic cell death at 12, 18, and 24 h postinduction. Activation of caspase-3 was evident in induced cells and cell death could be inhibited by the addition of a caspase-3-specific inhibitor. Overexpression of the myeloma-associated oncogene FGFR3, which upregulates bcl-x(L), delayed but did not prevent caspase-3-mediated killing. B9BM-C3 cells formed tumors after subcutaneous injection in mice. Early treatment with RU486 eradicated tumors; however, rechallenge of treated mice failed to demonstrate evidence of immunoprotection. These results indicate that therapeutic attempts to induce caspase-3 in malignant cells may prove useful in the treatment of bcl-x(L)-expressing tumors.

Molecular Target Characterization and Antimyeloma Activity of the Novel, Insulin-like Growth Factor 1 Receptor Inhibitor, GTx-134

Purpose: Therapeutic strategies that target insulin-like growth factor 1 receptor (IGF-1R) hold promise in a wide variety of cancers including multiple myeloma (MM). In this study, we describe GTx-134, a novel small-molecule inhibitor of IGF-1R and insulin receptor (IR) and characterized its antitumor activity in preclinical models of MM. Experimental Design: The activity of GTx-134 as a single agent and in combination was tested in MM cell lines and primary patient samples. Downstream effector proteins and correlation with apoptosis was evaluated. Cytotoxcity in bone marrow stroma coculture experiments was assessed. Finally, the in vivo efficacy was evaluated in a human myeloma xenograft model. Results: GTx-134 inhibited the growth of 10 of 14 myeloma cell lines (<5 μmol/L) and induced apoptosis. Sensitivity to GTx-134 correlated with IGF-1R signal inhibition. Expression of MDR-1 and CD45 were associated with resistance to GTx-134. Coculture with insulin-growth factor-1 (IGF-1) or adherence to bone marrow stroma conferred modest resistance, but did not overcome GTx-134–induced cytotoxicity. GTx-134 showed in vitro synergies when combined with dexamethasone or lenalidomide. Further, GTx-134 enhanced the activity of PD173074, a fibroblast growth factor receptor 3 (FGFR3) inhibitor, against t(4;14) myeloma cells. Therapeutic efficacy of GTx-134 was shown against primary cells and xenograft tumors. Although dysregulation of glucose homeostasis was observed in GTx-134–treated mice, impairment of glucose tolerance was modest. Conclusions: These studies support the potential therapeutic efficacy of GTx-134 in MM. Further, they provide a rationale for clinical application in combination with established antimyeloma treatments and novel targeted therapies. Clin Cancer Res; 17(14); 4693–704. ©2011 AACR.

Spontaneous Remission in a Patient With t(4;14) Translocation Multiple Myeloma

A 60-year-old woman was found to have a high erythrocyte sedimentation rate (121 mm/h) and low hemoglobin (113 g/L) in December 2005. Further work-up led to the diagnosis of DurieSalmon stage 1A/International Staging System stage I multiple myeloma (MM) based on the following: (1) a protein electrophoresis showed immunoglobulin (Ig) G, 14.0 g/L; IgA, 30.3 g/L; and IgM, 0.37 g/L; with a major spike in the beta region identified as an IgA-kappa paraprotein (Fig 1); (2) a bone marrow biopsy revealed 20% to 30% infiltration with atypical plasma cells, kappa restricted (Fig 2). A metastatic x-rays survey showed osteopenia throughout the axial skeleton with multiple subtle lucencies in the proximal humeral diaphyses bilaterally. A 24-hour urine collection showed 0.05 g/L of proteinuria with no Bence-Jones protein excretion. Peripheral blood counts revealed a hemoglobin level of 113 g/L (mean corpuscular volume, 90 fL), platelets at 281 10/L, and WBC of 6.8 10/L with 4.2 absolute neutrophil count. Electrolytes and calcium were within normal limits, but creatinine was slightly elevated at 107 mol/L (normal, 99 mol/L). 2-microglobulin, C-reactive protein, and albumin were 219 nmol/L (normal, 219 nmol/L), 4 mg/L (normal, 12 mg/L), and 36 g/L (range, 36 to 50 g/L), respectively. Further analysis of the malignant plasma cells by fluorescent in situ hybridization coupled to cytoplasmic staining of specific Ig revealed them to be positive for t(4;14) (Fig 3, white arrows). IgH-MMSET fusion transcripts were also detected by reverse transcriptase polymerase chain reaction, confirming the presence of t(4;14) cells in the specimen (data not shown). The plasma cells expressed FGFR3 by flow cytometry (data not shown). In addition, in vitro exposure of bone marrow mononuclear cells from this patient to XL999, a selective inhibitor of FGFR3 preferentially induced apoptosis of the CD138 cell fraction. Array comparative genomic hybridization revealed an amplification of the der(4)t(4;14) chromosome, thus explaining the three fusion signals seen by fluorescent in situ hybridization. Array comparative genomic hybridization also defined a hyperdiploid karyotype with trisomies of chromosomes 3, 4, 9, 18, and 19, and monosomy 13, as well as a focal deletion on chromosome X containing CYLC1, RPS6KA6, and SATL1—three genes that have been implicated in the pathogenesis of cancer (Fig 4). No active therapy was recommended apart from monthly intravenous Protein Electrophoresis

Abstract C246: SH-4-54, a novel small-molecule inhibitor of STAT3, demonstrates significant anti-tumor activity against multiple myeloma.

Aberrant STAT3 signaling is prevalent in hematologic malignancies such as Multiple Myeloma (MM) and is recognized as a master regulator of tumor processes such as survival, angiogenesis, and drug resistance. Significant efforts have therefore focused on therapeutic targeting of STAT3 as an anti-cancer strategy. Based on rational structure-based design, we present pre-clinical findings for a newly developed small molecule STAT3 inhibitor, SH-4-54. SH-4-54 was designed to block the SH2 domain of STAT3 by mimicking its native phosphopeptide binding sequence and in turn prevent phosphorylation (activation) and disrupt transcriptionally active STAT3:STAT3 dimerization. SH-4-54 has been shown to have a KD for STAT3 protein of 300 nM by surface plasmon resonance and inhibits STAT3 SH2 domain-phosphopeptide interactions as assessed by a fluorescence polarization assay. The anti-MM activity of SH-4-54 was assessed using MTT assays against a panel of molecularly heterogeneous human myeloma cell lines (HMCLs), revealing potent and broad effects on cell viability with 10/15 HMCLs displaying IC50 values < 10 μM. Moreover, SH-4-54 induced apoptotic responses in HMCLs as evidenced by increased Annexin V staining. Promising results were observed for the effects of SH-4-54 on primary patient-derived myeloma cells. Using flow cytometric analysis of CD138 as a marker of myeloma cells and Annexin V staining, SH-4-54 was found to significantly reduce the percentage of CD138+/Annexin V negative, viable myeloma cells, while displaying little to no toxicity against the non-malignant (CD138−) cell fraction. Supporting the predicted molecular mechanisms of SH-4-54 activity, immunoblot analysis revealed that SH-4-54 inhibits constitutive STAT3 phosphorylation, but does not alter STAT3 protein levels. Furthermore, using HMCLs engineered to express a STAT3-driven luciferase reporter construct, SH-4-54 was found to significantly reduce STAT3 transcriptional activity, and consistently induce a reduction in proteins that encode STAT3 target genes such as c-Myc. Taken together, our results highlight the promising therapeutic potential of SH-4-54 and support the continued development of targeted therapies for MM with a focus on aberrant STAT signaling. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C246. Citation Format: Zhi Hua Li, Sina Haftchenary, Danielle Croucher, Patrick T. Gunning, Suzanne Trudel. SH-4-54, a novel small-molecule inhibitor of STAT3, demonstrates significant anti-tumor activity against multiple myeloma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C246.