Novel Selective Orally Bioavailable Small Molecule PAK4 Allosteric Modulators (PAMs) Display Anti-Tumor Activity in Vitro and in Vivo in Hematological Malignancies

Abstract Introduction: Many hematological cancers have been successfully treated through identification of specialized targets in each specific tumor subtype (e.g. BTK inhibition in NHL or proteasome inhibition in multiple myeloma). The p21-Activated Kinase 4 (PAK4) is critical to cellular signaling and may represent a new target for therapy in many hematologic malignancies. PAK4 is a member of the PAK family of proteins that regulate cell survival, cell division and apoptosis. The six members of the PAK family are divided into two groups; Group I (PAK1, 2, 3) and Group II (PAK4, 5, 6), based upon their sequence homology and regulatory mechanisms. PAK4 is a member of the group II family of PAKs and is amplified or mutated in many cancer types. PAK4 is also a key downstream effector of the K-Ras pathway. Methods: Flow cytometry and CellTiter AQueous One (MTS) assays were used to determine compound effects on cell cycle distribution, proliferation and viability. Immunoblots were used to measure effects of compounds on protein steady state levels and phosphorylation. The T-cell ALL cell line, MOLT-4, and the mantle cell lymphoma cell line, Z-138, were used in xenograft models in mice to test the in vivo efficacy of these compounds. Results: We have identified selective, orally bioavailable, small molecule PAK4 allosteric modulators {PAMs; e.g. KPT-8752 (mw: 585.6), KPT-9274 (mw: 610.6), and KPT-9331 (mw: 628.6)} which demonstrated selective anti-tumor activity in a variety of hematological cancer cell lines (IC50 values = 0.005 – 1 mM). Treatment of cancer cells with these small molecules resulted in the reduction of PAK4 steady state levels and reduced phosphorylation of key growth signaling proteins such as Akt, β-catenin, cofilin, p21, and cyclin D1. There was a measurable increase in phospho-AMPK indicative of autophagy and stress. These allosteric modulators induced apoptosis through the activation of caspases 3 and 8 and subsequent cleavage of PARP. In MOLT-4 and Z-138 xenograft mouse models, daily treatment with oral PAMs resulted in near elimination of small (100 mm3) and large (800 mm3) tumors in the absence of any clinical signs of toxicity within the animals. Additional cell line and primary tumor models are currently being explored. Conclusions: PAK4 represents a novel anti-cancer target as a major downstream effector of the Ras oncogene. We have identified selective, orally-bioavailable small molecule PAK4 allosteric modulators which induce potent cytotoxicity in multiple leukemia and lymphoma cell lines with minimal toxicity to normal cell in vitro and clear anti-tumor activity with excellent tolerability in in vivo models of hematological cancers. These compounds inactivate PAK4 by directly inducing PAK4 destabilization. This represents a novel mechanism of the protein kinase inactivation involving degradation of PAK4 rather than direct inhibition of the kinase activity. Based on the in vitro and in vivo activity, these PAK4 allosteric modulators show promising results for the treatment of a wide variety of hematological cancers. Disclosures Senapedis: Karyopharm: Employment. George:Karyopharm: Employment. McCauley:Karyopharm Therapeutics: Employment, Equity Ownership. Ellis:Karyopharm: Employment. Crochiere:Karyopharm: Employment. Savona:Karyopharm: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Shacham:Karyopharm Therapeutics: Employment. Landesman:Karyopharm Therapeutics: Employment. Baloglu:Karyopharm: Employment.

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TTI-281, an Orally Bioavailable Bromodomain and Extraterminal Domain (BET) Inhibitor, Is a Promising Candidate for the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.2095.2095 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2095-2095

Author(s):

Zezhou Wang ◽

Jaehyun Choi ◽

Peter Dove ◽

Chunlei Wang ◽

Aaron D. Schimmer ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Therapeutic Agents ◽

Vehicle Control ◽

Advisory Committees ◽

Bet Inhibitor ◽

Low Toxicity ◽

Orally Bioavailable

Abstract Although recent advances in the development of multiple myeloma (MM) therapies such as proteasome inhibitors and immunomodulatory agents have improved patient outcomes, MM remains incurable. Additional therapeutic agents with high efficacy, low toxicity and the convenience of oral administration are in high demand. BET inhibitors, such as JQ-1, have been considered as potential therapeutic agents for MM. In the present study, we report that TTI-281, an orally bioavailable BET inhibitor, displays anti-MM activity with a low toxicity profile in preclinical studies. First, TTI-281 was tested for binding and anti-tumor activity in vitro. BROMOscan and AlphaScreen assays demonstrated that TTI-281 bound to bromodomains of BRD2/BRD3/BRD4 with Kd values less than 10 nM. In MTS assays, TTI-281 inhibited the growth of MM cell lines (MM.1s, NCIH929, and RPMI-8826) with cell growth-inhibition (IC50) values less than 300 nM. Next, in vitro ADME screening and in vivo PK studies were conducted. Permeability assays using murine gastrointestinal epithelial cells indicated that TTI-281 had good permeability with little efflux liability (efflux ratio <1), suggesting favorable properties for oral absorption. Indeed, TTI-281 displayed excellent oral bioavailability in both mice and rats (93.1% and 91.8%, respectively). In addition, TTI-281 did not interfere with the metabolism of representative CYP isozyme substrates at concentrations up to 50 μM in pooled human liver microsomes. Data also suggested minimal potential for drug-drug interactions, allowing for the possible combination with first-line therapy to improve therapeutic and survival outcomes. Finally, TTI-281 was tested for anti-myeloma efficacy and tolerability in vivo. NOD-SCID mice (n=10/group) subcutaneously engrafted with the human myeloma cell line MM.1S were treated orally once daily for 21 days with different doses of TTI-281, vehicle control or the benchmark drug carfilzomib. TTI-281 reduced tumor growth in a dose-dependent manner in this MM xenograft model. At 30 mg/kg/day, TTI-281 led to a statistically significant decrease in tumor growth compared with the vehicle control and carfilzomib (reduced tumor volume: 67% after TTI-281 treatment vs 33% after carfilzomib treatment, p<0.0003). Furthermore, TTI-281 treatment was well tolerated, with no effect on body weight or other obvious toxicity. In summary, our preclinical data suggest that the orally available BET inhibitor TTI-281 has an excellent efficacy and safety profile, highlighting its potential as a promising drug candidate for myeloma therapy. Disclosures Wang: Trillium Therapeutics: Employment, Patents & Royalties. Choi:Trillium Therapeutics: Employment. Dove:Trillium Therapeutics: Employment, Patents & Royalties. Wang:Trillium Therapeutics: Employment. Schimmer:Novartis: Honoraria. Petrova:Trillium Therapeutics Inc: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Uger:Trillium Therapeutics: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Slassi:Trillium Therapeutics: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

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TNFR2 As a Target to Improve CD19-Directed CART Cell Fitness and Antitumor Activity in Large B Cell Lymphoma

Blood ◽

10.1182/blood-2021-148591 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 901-901

Author(s):

Claudia Manriquez Roman ◽

Michelle J. Cox ◽

Reona Sakemura ◽

Kun Yun ◽

Mohamad M. Adada ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Cell Activation ◽

B Cell Lymphoma ◽

Death Receptors ◽

Target Cells ◽

Advisory Committees ◽

Anti Tumor Activity

Abstract Introduction: It has become increasingly apparent that chimeric antigen receptor T (CART) cell activation and differentiation level is an important determinant of CART cell fate and response to therapy. In this study, we aimed to 1) measure levels of activation-induced surface death receptors and ligands on CART cells; 2) investigate how CART cell activation could impact their fitness and clinical responses, and 3) identify cell-based targets to modulate CART cell activation, apoptosis, and cytotoxicity to improve anti-tumor activity. Methods: We performed flow cytometric studies on ex-vivo stimulated, clinically annotated CART products of patients with large B cell lymphoma from the pivotal ZUMA-1 clinical trial that led to FDA-approved Axicabtagene ciloleucel (Axi-Cel). We investigated possible correlations of a number of surface death receptors and ligands with T cell differentiation status and post-infusion CART cell expansion, utilizing samples from ZUMA-1 patients who achieved a complete response as a best outcome ('responders') vs patients who achieved stable or progressive disease('non-responders'). CART cell effector functions in vitro were measured, and CART apoptosis was assessed using Annexin V. For in vitro and in vivo functional studies, we used CART19 generated from healthy donors (HD CART19) as indicated in the specific experiment. CRISPR/Cas9 was employed during CART cell production to disrupt specific genes. A xenograft model of lymphoma was used to investigate the in vivo antitumor activity of CART19. Results: Following an ex vivo stimulation of Axi-Cel products with CD19 + target cells, we observed upregulation of death receptors and ligands in CART19 from non-responders, compared to responders. We also observed a possible association between such upregulated surface markers with CART cell differentiation as measured by CCR7 expression. In an extended in vitro co-culture assay, where HD CART19 cells were repeatedly stimulated through the CAR, we found that tumor necrosis factor α receptor 2 (TNFR2), unlike other death receptors and ligands, was persistently elevated, suggesting a possible role for TNFR2 in long-term antigen-dependent CART19 dysfunction (Figure 1A). We further found that HD CART19 upregulate TNFR2, but not TNFR1, upon CAR stimulation (Figure 1B). While non-specific TCR activation (CD3 stimulation) of HD CART19 cells protected them from activation-induced apoptosis, antigen-specific activation through the CAR resulted in significant initiation of apoptosis within 2 hours of stimulation (Figure 1C). Having identified a possible association between TNFR2 and CART19 dysfunction, we aimed to study the impact of TNFR2 knockout on HD CART19 functions. Using CRISPR/Cas9 during CART cell manufacturing, we generated TNFR2 k/o HD CART19 cells with a knockout efficiency of around 50%, where the expression levels of TNFR2 in activated CART19 cells were reduced, compared to control CART19 cells (with non-targeting gRNA CRISPR/Cas9, Figure 1D). TNFR2 k/o CART19 cells demonstrated reduced early activation surface markers compared to control CART19, as measured by CD25 and CD69 surface expression (Figure 1E), reduced apoptosis initiation as measured by the Annexin V assay (Figure 1F), and enhanced antigen-specific proliferation and cytotoxicity (Figure 1G). Finally, in an in vivo xenograft model of CD19 + lymphoma, TNFR2 k/o CART19 resulted in enhanced CART cell expansion and anti-tumor activity (Figure 1H). Conclusions: Our results indicate that TNFR2 plays a role in early activation and apoptosis initiation of CART19 following CAR stimulation with CD19 + target cells and present TNFR2 knockout as a strategy to enhance CART19 anti-tumor activity. Figure 1 Figure 1. Disclosures Cox: Humanigen: Patents & Royalties. Sakemura: Humanigen: Patents & Royalties. Ding: Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; MEI Pharma: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Genentech: Research Funding; Behring: Membership on an entity's Board of Directors or advisory committees; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; TG Therapeutics: Research Funding; Tolero Pharmaceuticals: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees. Scholler: Kite: Current Employment. Bot: Kite, a Gilead Company: Current Employment; Gilead Sciences: Consultancy, Current equity holder in publicly-traded company, Other: Travel support. Mattie: Kite: Current Employment. Kim: Gilead Sciences: Current equity holder in publicly-traded company; Kite, a Gilead Company: Current Employment. Filosto: Kite, a Gilead Company: Current Employment; Tusk Therapeutics: Patents & Royalties: or other intellecular property; Gilead Sciences: Other: stock or other ownership . Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding.

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Vesicular Stomatitis Virus (VSV) Engineered to Express CD19 Stimulates Anti-CD19 Chimeric Antigen Receptor Modified T Cells and Promotes Their Anti-Tumor Effects

Blood ◽

10.1182/blood-2020-139647 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 30-31

Author(s):

Reona Sakemura ◽

Elizabeth C. Eckert ◽

Sydney B. Crotts ◽

Linh Pham ◽

Elizabeth L. Siegler ◽

...

Keyword(s):

Cancer Cells ◽

Board Of Directors ◽

Immune Cells ◽

Research Funding ◽

Leukemia Cell ◽

Leukemia Cell Line ◽

Advisory Committees ◽

Anti Tumor Activity

Although CD19-directed chimeric antigen receptor T cell (CART19) therapy is highly effective and was FDA approved for certain B-cell malignancies, most patients relapse after CART infusion within the first 1-2 years due to inadequate CART expansion in vivo. Vesicular stomatitis virus (VSV) has the ability to infect and lyse cancer cells. Clinical trials of VSV oncolytic therapy indicate that VSV efficiently infects cancer cells as well as innate immune cells. Therefore, we hypothesized that in patients who achieve suboptimal response to CART19, VSV engineered to express CD19 will augment anti-tumor activity through 1) direct lysis of cancer cells and 2) infecting cancer cells and innate immune cells with CD19 to further stimulate CART19. To test our hypothesis, human CD19 or GFP (control) was engineered between the glycoprotein and large-protein (Fig.1A) in a modified VSV backbone. A matrix inactivating mutation (M51R) rendered it incapable of suppressing anti-viral reactions of infected targets, potentially promoting its immunogenicity. First, we tested the anti-tumor activity of VSV-CD19 and VSV-GFP against the luciferase (luc)+CD19+ acute lymphoblastic leukemia cell line NALM6 and the luc+CD19- acute myeloid leukemia cell line MOLM13. VSV-CD19 and VSV-GFP successfully lysed NALM6 (Fig.1B) or MOLM13, both in vitro and in vivo (data not shown). Next, we investigated the efficiency of VSV-CD19 in infecting tumor and immune cells. 24 hours after exposure to VSV-CD19 or VSV-GFP, we analyzed the surface expression of CD19 on MOLM13 and revealed efficient CD19 delivery (Fig.1C). Next, we assessed VSV infection of peripheral blood mononuclear cells (PBMCs) from healthy donors (HDs). Freshly isolated HD PBMCs were infected with VSV-CD19 for 6 hours and subsequently assessed for CD19 expression. Consistent with findings from clinical trials, VSV-CD19 selectively infected and induced CD19 expression on monocytes while other cells were not affected (Fig.1D). To exclude potential toxicities against CART19, we co-cultured CART19 with VSV-CD19 or VSV-GFP using second-generation 4-1BB costimulated CART19. Both VSV-CD19 and VSV-GFP did not infect CART19 as evident by preservation of CART19 viability and lack of CD19 or GFP expression (Fig.1E). Having demonstrated that VSV-CD19 specifically delivered CD19 to monocytes, we next tested whether the infected monocytes stimulated CART19. VSV-CD19 infected monocytes induced potent antigen-specific proliferation of CART19 (Fig.1F) and resulted in enhanced anti-tumor activity against luc+NALM6 in vitro (Fig.1G). Next, we aimed to confirm these findings in vivo. We generated luc+CART19 to track CART19 expansion in vivo. Freshly isolated HD monocytes were infected with VSV-CD19 ex vivo. After 4 hours, VSV-CD19 was washed away and immunocompromised NSG mice were intravenously injected with VSV-CD19 infectedmonocytes. After 24 hours, 3.5x106 of luc+untransduced T cells (UTD) or luc+CART19 were injected intravenously. The T cell expansion was assessed by bioluminescence imaging (BLI). VSV-CD19 infected monocytes specifically stimulated and expanded CART19 (Fig.1H). Finally, we tested whether VSV-CD19 can stimulate and rescue suboptimal anti-tumor effects of CART19 in vivo using a NALM6 relapsed model. Here, 1x106 luc+NALM6 were injected intravenously into NSG mice on day -6. At day -1, mice were imaged and randomized according to tumor burden to receive 1x106 UTD or CART19 on day 0. Subsequently, at day 4, mice were re-imaged and randomized. At day 5, HD monocytes were injected intravenously. Three hours after administering monocytes, mice received 1x107 VSV-CD19 or VSV-GFP (Fig.1I). BLI revealed that CART19 plusVSV-CD19 showed better tumor control than CART19 monotherapy or CART19 plus VSV-GFP (Fig.1J-K). Furthermore, CART19 plus VSV-CD19 exhibited long-term survival (Fig.1L). In summary, VSV-CD19 not only demonstrated direct anti-tumor effects but also specifically delivered CD19 to monocytes and tumor cells, thereby re-stimulating and enhancing the anti-tumor activity of CART19. This work provides a rationale to study VSV-CD19 in patients who demonstrate only suboptimal response to CART19. This approach could also be applied to augment CART therapy in other tumors. Figure 1 Disclosures Sakemura: Humanigen: Patents & Royalties. Eckert:Genentech: Current Employment. Cox:Humanigen: Patents & Royalties. Parikh:Ascentage Pharma: Research Funding; GlaxoSmithKline: Honoraria; Verastem Oncology: Honoraria; MorphoSys: Research Funding; Genentech: Honoraria; Pharmacyclics: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Merck: Research Funding; Janssen: Honoraria, Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Honoraria, Research Funding. Kay:Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding. Peng:Imanis: Other: Equity Ownership. Russell:Imanis: Other: Equity Ownership. Kenderian:Mettaforge: Patents & Royalties; Humanigen: Consultancy, Patents & Royalties, Research Funding; Lentigen: Research Funding; Torque: Consultancy; Novartis: Patents & Royalties, Research Funding; Kite: Research Funding; Gilead: Research Funding; Juno: Research Funding; BMS: Research Funding; Tolero: Research Funding; Sunesis: Research Funding; MorphoSys: Research Funding.

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Dual Targeting of Ph+ ALL with Dasatinib and ABT-199 (Venetoclax)

Blood ◽

10.1182/blood.v126.23.1329.1329 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1329-1329

Author(s):

Jessica Leonard ◽

Joelle Rowley ◽

Brandon Hayes-Lattin ◽

Jeffrey W. Tyner ◽

Marc Loriaux ◽

...

Keyword(s):

Older Adults ◽

Cell Line ◽

Board Of Directors ◽

Research Funding ◽

Mononuclear Cells ◽

Annexin V ◽

Advisory Committees ◽

Equity Ownership

Abstract Introduction: Treatment of adult Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL) remains a challenge. While the addition of the targeted tyrosine kinase inhibitors (TKI) to standard cytotoxic therapy has greatly improved upfront treatment, treatment related mortality in older adults remains high. A novel induction regimen combines the targeted dual Abl/Src TKI Dasatinib (Sprycel, BMS) with a corticosteroid. After the first 21 days of induction the corticosteroids are tapered due to significant toxicities, particularly in older adults. Unfortunately, remaining on TKI monotherapy renders patients susceptible to the development of TKI resistance and thus identifying targeted agents that could enhance the activity of TKIs is urgently needed. Recently a novel and selective inhibitor of BCL-2, ABT-199 (Venetoclax, AbbVie) has shown impressive activity against other lymphoid malignancies including CLL and NHL. Here we describe the pre-clinical and in vivo efficacy of ABT-199 in combination with dasatinib in Ph+ ALL and propose its potential use in future clinical trials. Methods: Drug efficacy in vitro was determined using the Ph+ ALL cell line SupB15, primary Ph+ ALL sample (12-149), the dasatinib sensitive Pre-B ALL cell line RCH and the CML cell line K562. Cells were treated with dasatinib, ABT199 or in combination for 72 hours. Cell viability was assessed with the colorimetric MTS assay and apoptosis was assessed with annexin V staining. Expression of the BCL family proteins BCL-2 and MCL-1 were assessed via immunoblot. Immunodeficient NSG mice were injected with 12-149, then one week later treated with vehicle, 5 mg/kg dasatinib, 5 mg/kg ABT-199, or the combination daily for 5 days each week. Peripheral blood was obtained every 1-2 weeks to assess for engraftment as defined by the presence of >10% human CD45+ cells in the peripheral blood. Once engrafted, mice were euthanized and examined. Mononuclear cells were extracted and assessed for BCL2 and MCL1 expression. Statistical methods were performed using Calcusyn and PRISM. Results: Susceptibility to BCL2 inhibition: Of the dasatinib sensitive cells tested, SupB15 and 12-149 cells were susceptible to ABT-199 while RCH and K562 cells were not. The ALL cells expressed BCL-2 while the CML cell line expressed BCLx. SupB15 expressed low levels of the antiapoptotic protein MCL1 while RCH cells had relatively higher levels. siRNA of MCL-1 rendered the RCH cells sensitive to inhibition by ABT-199. In SupB15 cells, treatment with ABT-199 alone led to upregulation of MCL-1 at 24h which was prevented by the combination of dasatinib + ABT199. Synergy in Ph+ ALL: The calculated IC50 of dasatinib and ABT199 in SupB15 were 8.8nM and 5.9nM, respectively. The IC50 of equimolar combination was 0.42nM, and synergistic with combination index (CI) values between 0.15 and 0.49. Primary Ph+ ALL xenograft cells showed a similar pattern of synergy to the dasatinib + ABT199 combination. Combination treatment also greatly increased apoptosis as measured by Annexin V staining. Xenograft Studies: Animals were treated with a ten-fold lower dose of dasatinib and ABT199 from prior published data. There was no significant difference in time to engraftment or disease burden between vehicle or single agent ABT-199. In contrast, less than one half of the animals treated with dasatinib engrafted by 90 days while none of the animals treated with both dasatinib and ABT-199 engrafted. Most intriguing was the decrease in disease burden as measured by splenic size in the combination group compared to all other groups (P<0.0001, one-way ANOVA). Analysis of BCL-2 family proteins from mononuclear cells isolated from untreated animals confirmed upregulation of BCL-2 and relatively low levels of MCL-1. Animals treated with ABT-199 had greatly upregulated levels of MCL-1, while those treated with dasatinib or the combination did not. Conclusions: The combination of ABT-199 with dasatinib synergistically targets Ph+ ALL cells both in vitro and in vivo, laying the foundation for further evaluation in vivo for adult Ph+ ALL. As demonstrated by others, malignancies that are particularly susceptible to BCL targeting are those which display high BCL-2 expression and a low MCL-1: BCL-2 ratio. Combined targeted therapies may offer the potential for greater and longer responses without the morbidity associated with cytotoxic chemotherapy, particularly in older adults. Disclosures Tyner: Aptose Biosciences: Research Funding; Janssen Pharmaceuticals: Research Funding; Incyte: Research Funding; Array Biopharma: Research Funding; Constellation Pharmaceuticals: Research Funding. Druker:Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; McGraw Hill: Patents & Royalties; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Millipore: Patents & Royalties; Fred Hutchinson Cancer Research Center: Research Funding; Novartis Pharmaceuticals: Research Funding; Sage Bionetworks: Research Funding; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Henry Stewart Talks: Patents & Royalties; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotide Pharmaceuticals: Research Funding; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy.

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Mechanism Study of Matrix Metalloproteinase 13 Effects on Osteoclast Activation and Lytic Bone Lesions in Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.3009.3009 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3009-3009

Author(s):

Jing Fu ◽

Shirong Li ◽

G. David Roodman ◽

Markus Mapara ◽

Stephen J. Weiss ◽

...

Keyword(s):

Cell Proliferation ◽

Enzymatic Activity ◽

Cell Line ◽

Board Of Directors ◽

Underlying Mechanism ◽

Advisory Committees ◽

Matrix Metalloproteinase 13 ◽

Nf Kappab

Abstract Background : We have recently shown that Matrix metalloproteinase-13 (MMP-13) is highly expressed in human multiple myeloma (MM) cells and MMP-13 increases osteoclast (OCL) fusion and bone resorption independently of its metalloproteinase activity. In a mouse model of MM bone disease, knockdown (KD) of MMP-13 in MM cells inhibited development of bone lytic lesions, suggesting that MMP-13 is a potential therapeutic target for the treatment of MM bone disease (MMBD). However, the effects of MMP-13 on the MM bone marrow (BM) niche and underlying mechanism by which MMP-13 induces OCL fusion remains undefined. Methods and Results: To delineate the effects of MMP-13 on MM BM microenvironment, we investigated the effects of MMP-13 on cell proliferation and differentiation of pre-OCL, pre-osteoblast and MM cells. WT or Mmp-13-/- mouse CD11b+ BM cells were cultured with or without MMP-13 for 3 days and pre-OCL proliferation was evaluated by WST-1 assay. Neither MMP-13 treatment nor Mmp-13 deficiency affected pre-OCL proliferation, indicating MMP-13 induces OCL activation rather by promoting cell fusion than by affecting cell proliferation. To test the effects of MMP-13 on osteoblast (OB) differentiation and mineralization, mouse pre-OB cell line MC3T3-E1 and primary pre-OB cells from WT or Mmp-13-/- mice BM were cultured with or without MMP-13 for 7-14d. Neither exogenous MMP-13 nor Mmp-13 knockout affected OB differentiation or mineralization in vitro. Consistent with the in vitro observation, our Rag2-/- mouse 5TGM1 MMBD model provided in vivo evidence that knockdown of MMP-13 in MM cells does not affect MM induced OB inhibition or impairment of bone formation. Further, the effects of MMP-13 on MM cell proliferation were also assessed both in vitro and in vivo. Treatment of human MM cell lines RPMI-8266, OPM2 and MM.1S, as well as mouse MM cell line 5TGM1 with MMP-13 for up to 3 days did not induce in vitro cell proliferation. Similarly, MM cell sensitivity against anti-MM agents bortezomib and pomalidomide was not affected by MMP-13 in vitro. But, MMP-13 KD in mouse 5TGM1 MM cell line caused significantly less in vivo tumor burden in Rag2-/- intratibial MMBD model compared to 5TGM1 empty vector control cells, which we speculate occurs as a consequence of effects secondary to decreased OCL activation within the bone microenvironment following MMP-13 silencing. Our previous experiments showed that MMP-13 induces OCL fusion independent of its enzymatic activity, however the underlying mechanism is unknown. To further analyze the mechanism of action immunofluorescence staining and cell fraction-immunoblotting assay demonstrated that extracellular MMP-13 undergoes cell surface binding and subsequent active intracellular translocation into the perinuclear area, further supporting the enzymatic activity independent mechanism of MMP-13 on OCL induction. Furthermore, analysis of MMP-13-induced signaling pathways mediating osteoclastogenesis revealed that MMP-13 activates multiple cell signaling pathways including MAPK, PI3K and NF-kappaB independent of its enzymatic activity. Interestingly, the noncanonical NF-kappaB pathway is selectively activated by either MMP-13 WT or an E223A catalytically-inactive mutant, but not any by any other MMPs or MMP-13 truncated non-functional mutants, indicating that signaling through the noncanonical NF-kappaB pathway in OCL is specific for MMP-13 and may play a role in MMP-13-driven OCL fusion. Further studies are underway to screen for MMP-13 receptors and binding partners in an effort to delineate the processes underlying MMP-13 signaling. Conclusion: Our results demonstrate that MM produced MMP-13 induces OCL activation and bone resorption without affecting OB inhibition and bone formation impairment as well as anti-neoplastic response of MM cells. MMP-13 silencing in MM protects bone structure, and inhibits MM progression in vivo, which may be secondary to decreased OCL activation. MMP-13 specifically acts on OCL fusion. Signaling study showed noncanonical NF-kappaB signaling is selectively activated by MMP-13 independently of its enzymatic activity, and may be critical for MMP-13-induced OCL fusion and activation. Further screening the potential cellular receptor and intracellular transportation mechanism of MMP-13 in OCL is ongoing. Disclosures Roodman: Amgen: Consultancy; Eli Lilly: Research Funding. Lentzsch:Axiom: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

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SOMCL-863, a novel, selective and orally bioavailable small-molecule c-Met inhibitor, exhibits antitumor activity both in vitro and in vivo

Cancer Letters ◽

10.1016/j.canlet.2014.05.012 ◽

2014 ◽

Vol 351 (1) ◽

pp. 143-150 ◽

Cited By ~ 8

Author(s):

Lu Wang ◽

Jing Ai ◽

Yanyan Shen ◽

Haotian Zhang ◽

Xia Peng ◽

...

Keyword(s):

Antitumor Activity ◽

Small Molecule ◽

Met Inhibitor ◽

Orally Bioavailable

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Preclinical Evaluation of A Potent 2nd Generation Small-Molecule Hsp90 Inhibitor STA-9090 In Hematological Cell Lines

Blood ◽

10.1182/blood.v116.21.2899.2899 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2899-2899

Author(s):

Weiwen Ying ◽

David Proia ◽

Suqin He ◽

Jim Sang ◽

Kevin Foley ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Small Molecule ◽

First Generation ◽

Cell Lymphoma ◽

Hsp90 Inhibitor ◽

Phase 2 ◽

Hsp90 Inhibitors

Abstract Abstract 2899 STA-9090 is a potent, second generation, small-molecule Hsp90 inhibitor, with a chemical structure unrelated to the first-generation, ansamycin family of Hsp90 inhibitors. In preclinical in vitro and in vivo studies, STA-9090 has shown potency up to 100 times greater than the first-generation Hsp90 inhibitors against a wide range of solid and hematological cancer types including those resistant to imatinib, sunitinib, erlotinib, and dasatinib. STA-9090 is currently being evaluated two Phase 1 and four Phase 2 trials (non-small cell lung, GIST, colon, and gastric) in solid tumor cancers; and two trials in hematologic cancers. Additional Phase 2 trials in several other indications are planned for 2H 2010. Inhibition of Hsp90 by STA-9090 results in the destabilization of a broad range of oncogenic kinases often overexpressed or mutated in hematological cancers. For example, the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressed in the anaplastic large cell lymphoma (ALCL) cell line Karpas 299, is degraded rapidly in the presence of STA-9090 in vitro, resulting in the loss of viability. Similar results were shown in other NPM-ALK driven ALCL cells including SU-DHL-1 and SR-786 with IC50 less than 20 nM. Stability of other kinases common to hematological malignancies, such as Bcr-Abl, FLT3 and c-Kit, were also shown to be highly sensitive to STA-9090, resulting in potent cell death of cell lines addicted to signaling by these kinases. In vivo, STA-9090 was highly effective in a subcutaneous xenograft model of diffuse large B-cell lymphoma SU-DHL-4 with resulting %T/C values of 26, 4, -90 and -93 when dosed at 25, 50, 75 and 100 mg twice per week, respectively. Importantly, 75 and 100 mg/kg STA-9090 dosed 2 times per week for a total of 3 weeks (150 and 200 mg/kg weekly) resulted in 25% and 50% of the animals in each group being free of tumors by the end of the study, respectively. MV4-11, an AML (FLT3ITD) cell line, turned out to be one of the most sensitive xenograft models to STA-9090 treatment. STA-9090 at 100 mg/kg or125mg/kg once weekly was highly efficacious with 37.5% of mice achieving tumor free with acceptable toxicity at the end of the 3-week treatment period. In conclusion, STA-9090 exhibits preferable biological profiles both in vitro and in vivo in treating hematological malignances. Clinical studies for using STA-9090 both once weekly and twice weekly are ongoing. Disclosures: Ying: Synta Pharmaceuticals: Employment. Proia:Synta Pharmaceuticals: Employment. He:Synta Pharmaceur: Employment. Sang:Synta Pharmaceuticals: Employment. Foley:Synta Pharmaceuticals: former employee. Du:Synta Pharmaceuticals: former employee. Blackman:Synta Pharmaceuticals: Employment. Wada:Synta Pharmaceuticals: Employment. Sun:Synta Pharmaceuticals: Employment. Koya:Synta Pharmaceuticals: Employment.

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A Novel Acanthoic Acid Analog NPI-1342 Blocks IκB Kinase-α and Trigger In Vitro and In Vivo cytotoxicity in Multiple Myeloma Cells

Blood ◽

10.1182/blood.v118.21.1841.1841 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1841-1841

Author(s):

Dharminder Chauhan ◽

Ajita V. Singh ◽

Arghya Ray ◽

Teru Hideshima ◽

Paul G. Richardson ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Board Of Directors ◽

Tumor Cells ◽

Cell Lines ◽

Inhibitory Effect ◽

Advisory Committees ◽

Acid Analog

Abstract Abstract 1841 Introduction: The dimeric Nuclear Factor-kappa B (NF-κB) transcription factor plays a key role during multiple myeloma (MM) cell adhesion-induced cytokine secretion in bone marrow stromal cells, which in turn triggers MM cell growth in a paracrine manner. NF-κB signaling pathway is mediated via canonical (IKK-α/IKK-β/NEMO-P50/65 or NF-κB1) and non-canonical (IKK-α/IKK-α/NIK-p52/RelB or NF-κB2) components. Prior studies have also linked constitutive activation of non-canonical NF-κB pathway to genetic abnormalities/mutation, allowing for an autocrine growth of MM cells. Other recent studies showed that constitutive NF-κB activity in tumor cells from MM patients renders these cells refractory to inhibition by bortezomib; and in fact, that bortezomib induces canonical NF-κB activity. These reports provided the impetus for the development of an agent with ability to modulate canonical and/or non-canonical NF-κB axis, allowing for a more robust and specific inhibition of NF-κB. Recent research and development efforts at Nereus Pharmaceuticals, Inc., have identified a novel small molecule acanthoic acid analog NPI-1342 as a potent NF-κB inhibitor. Here, we examined the effects of NPI-1342 on canonical versus non-canonical NF-κB signaling pathways, as well as its anti-tumor activity against MM cells using both in vitro and in vivo model systems. Methods: We utilized MM.1S, MM.1R, RPMI-8226, U266, KMS12PE, NCI-H929, OCI-MY5, LR5, Dox-40, OPM1, and OPM2 human MM cell lines, as well as purified tumor cells from patients with MM. Cell viability assays were performed using MTT and Trypan blue exclusion assays. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymology assays. Animal model studies were performed using the SCID-hu model, which recapitulates the human BM milieu in vivo. Results: We first examined the effects of NPI-1342 on lipopolysaccharides (LPS)-induced NF-κB activity. Results showed that NPI-1342 inhibits LPS-stimulated NF-κB activity in vitro, as measured by phosphorylation of IkBa. To determine whether NPI-1342 triggers a differential inhibitory effect on IKKβ versus IKKα, MM.1S MM cells were treated with NPI-1342 for 48 hours, and protein lysates were subjected to kinase activity assays. NPI-1342 blocked IKKα, but not IKKβ or IKKγ phosphorylation. We next assessed whether the inhibitory effect of NPI-1342 on NF-κB activity is associated with cytotoxicity in MM cells. We utilized a panel of MM cell lines: at least five of these have mutations of TRAF3 (MM.1S, MM.1R, DOX40 and U266); one has no known NF-κB mutations (OPM2), and one has amplification of NF-κB1 (OCI-MY5). Treatment of MM cell lines and primary patient (CD138 positive) MM cells for 48 hours significantly decreased their viability (IC50 range 15–20 μM) (P < 0.001; n=3) without affecting the viability of normal peripheral blood mononuclear cells, suggesting selective anti-MM activity and a favorable therapeutic index for NPI-1342. NPI-1342-induced a marked increase in Annexin V+ and PI- apoptotic cell population (P < 0.001, n=3). Mechanistic studies showed that NPI-1342-triggered apoptosis in MM cells is associated with activation of caspase-8, caspase-9, caspase-3, and PARP cleavage. We next examined the in vivo effects of NPI-1342 in human MM xenograft models. For these studies, we utilized the SCID-hu MM model, which recapitulates the human BM milieu in vivo. In this model, MM cells are injected directly into human bone chips implanted subcutaneously in SCID mice, and MM cell growth is assessed by serial measurements of circulating levels of soluble human IL-6R in mouse serum. Treatment of tumor-bearing mice with NPI-1342 (20 mg/kg intraperitoneally, QD1-5 for 2 weeks), but not vehicle alone, significantly inhibits MM tumor growth in these mice (10 mice each group; P = 0.004). The doses of NPI-1342 were well tolerated by the mice, without significant weight loss. Finally, immunostaining of implanted human bone showed robust apoptosis and blockade of NF-κB in mice treated with NPI-1342 versus vehicle alone. Conclusions: We demonstrate the efficacy of a novel small molecule inhibitor of NF-κB NPI-1342 in MM using both in vitro and in vivo models. NPI-1342 blocks NF-κB activity with a preferential inhibitory activity against IKK-α component of NF-κB signaling. Our preclinical studies support evaluation of NPI-1342 as a potential MM therapy. Disclosures: Hideshima: Acetylon: Consultancy. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Palladino:Nereus Pharmaceuticals, Inc: Employment, Equity Ownership. Anderson:Celgene: Consultancy; Millennium: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Acetylon:; Nereus Pharmaceuticals, Inc: Consultancy.

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Role Of B Cell Tolerance In PF4/Heparin Antibody Production

Blood ◽

10.1182/blood.v122.21.2396.2396 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2396-2396

Author(s):

Yongwei Zheng ◽

Alexander W Wang ◽

Mei Yu ◽

Anand Padmanabhan ◽

Benjamin E Tourdot ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Board Of Directors ◽

Cell Tolerance ◽

Inflammatory Stimulus ◽

Wild Type ◽

B Cell Tolerance ◽

Advisory Committees

Abstract Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that can cause fatal arterial or venous thrombosis/thromboembolism. Immune complexes consisting of heparin, platelet factor 4 (PF4) and PF4/heparin-reactive antibodies are central to the pathogenesis of HIT. However, heparin, a glycosoaminoglycan, and PF4 are normal body constituents and it is as yet unclear what triggers the initial induction of pathogenic antibodies. Here we described detection of B cells among peripheral blood mononuclear cells (PBMCs) from each of 9 healthy adults that produced PF4/heparin-specific IgM antibodies following in vitro stimulation with ubiquitous pro-inflammatory molecules containing unmethylated CpG dinucleotides derived from bacterial and viral DNA. PF4/heparin-specific IgM-generating B cells were present at a frequency of at least 0.03 to 1 per thousand B cells present in the PBMC population. Similarly, splenic B cells isolated from unmanipulated wild-type mice consistently produced PF4/heparin-reactive antibodies following in vitro stimulation with CpG. In addition, wild-type mice produced PF4/heparin-reactive antibodies upon in vivo challenge with CpG whereas unchallenged wild-type mice did not. These findings demonstrate that both humans and mice possess pre-existing, inactive and tolerant PF4/heparin-specific B cells. We suggest that tolerance can be broken by a strong inflammatory stimulus, leading to activation of these B cells and production of antibodies that recognize PF4/heparin in vitro and in vivo. Consistent with this concept, mice lacking protein kinase Cd (PKCd), a signaling molecule of the B-cell survival factor BAFF (B-cell activation factor), that are known to have breakdown of B-cell tolerance to self-antigens, spontaneously produced anti-PF4/heparin antibodies in the absence of an inflammatory stimulus. Taken together, these findings demonstrate that breakdown of tolerance can lead to PF4/heparin-specific antibody production and that B-cell tolerance plays an important role in HIT pathogenesis. Disclosures: White II: Bayer: Membership on an entity’s Board of Directors or advisory committees; CSL-Behring: Membership on an entity’s Board of Directors or advisory committees; NIH: Membership on an entity’s Board of Directors or advisory committees; Asklepios: Membership on an entity’s Board of Directors or advisory committees; Wyeth: Membership on an entity’s Board of Directors or advisory committees; Entegrion: Membership on an entity’s Board of Directors or advisory committees; Biogen: Membership on an entity’s Board of Directors or advisory committees; Baxter: Membership on an entity’s Board of Directors or advisory committees.

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Evaluation of IGHV Ultra-Deep Sequences for Activation-Induced Deaminase Characteristics in CLL Cells after T Cell Stimulation

Blood ◽

10.1182/blood.v122.21.2583.2583 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2583-2583

Author(s):

Charles C. Chu ◽

Piers E.M. Patten ◽

Thomas MacCarthy ◽

Xiao-Jie Yan ◽

Jacqueline C. Barrientos ◽

...

Keyword(s):

T Cell ◽

Board Of Directors ◽

Deep Sequencing ◽

Ex Vivo ◽

Advisory Committees ◽

Polymerase Eta ◽

Activation Induced Deaminase ◽

T Cell Help

Abstract Ultra-deep sequencing has revolutionized our ability to acquire large amounts of genetic data. We have applied this technology towards understanding the mutational process in B-cell chronic lymphocytic leukemia (CLL), which may be a key to understanding CLL pathogenesis. Acquisition of new cytogenetic aberrations and gene mutations in the CLL clone is associated with worse patient outcome. CLL is not unique in this aspect, as new somatic mutations and DNA rearrangements are also found during the evolution of other solid and liquid tumors. In many of these, activation-induced deaminase (AID), an enzyme normally expressed in germinal center B lymphocytes to induce IGHV-D-J mutations and isotype class switch recombination, is abnormally expressed. Its mutational activity, acting outside of the Ig loci, is implicated in the evolution to more aggressive disease. In CLL, the detection of leukemic cells expressing AID ex vivo correlates with significantly shorter patient survival. To test if AID mutational activity is functional in CLL cells and therefore could contribute to CLL evolution, we analyzed mutations in IGHV-D-J, the preferred substrate for AID. Because the rate of AID-induced mutation is low and only a small percentage of CLL cells express AID ex vivo, we used ultra-deep sequencing to analyze CLL cells that were activated under conditions that simulate the CLL microenvironment. Specifically, CLL cells were activated (1) in vitro by simulating the provision of T-cell help or (2) in vivo after adoptive transfer into alymphoid recipient mice, which requires the presence of T-cells for CLL cell growth. Each of these conditions induce AID in a large fraction of CLL cells. To analyze IGHV-D-J mutations, the specific CLL clone IGHV was amplified from cDNA obtained on day 0 or from the activated CLL samples using IGHV family-specific and IGHM primers to enable subsequent comparison of IGHV-D-J with IGHM mutation frequencies. Three unmutated IGHV CLL (U-CLL) and 3 mutated IGHV (>2% compared to germline) CLL (M-CLL) samples were sequenced with the Roche 454 FLX system, resulting in a total of 1,367,522 sequence reads. After using the Roche 454 algorithm to trim sequence reads, they were prepared using custom R scripts that separated 5’ IGHV and 5’ IGHM primer sequences, aligned sequences to the CLL clone IGHV-D-J rearrangement, and removed poor quality (<20) sequences, insertions, and deletions. Beginning at the 5’ end, the script also extracted blocks of sequences of the same length for day 0 and activated samples, which are required for subsequent analyses. After these preparations, the resulting 724,855 sequence blocks were subjected to clonal analyses with custom R scripts. The dominant CLL clone accounted for 94.5% (684,691) of the sequences. Subclone sequences occurring more than once were extracted. After comparison to day 0, new subclones could be identified in all samples after activation (3.22 – 28.70 new subclones / read bp *106). To evaluate AID mutational characteristics in new subclones, SHMTool (http://scb.aecom.yu.edu/shmtool) was employed to calculate mutation frequencies in IGHV-D-J relative to the IGHM constant region, at AID mutation hotspot sites (GYW or WRC), at AID mutation coldspot sites (SYC or GRS), at C/G base pairs, and at error-prone DNA polymerase eta repair hotspot sites (WA or TW). To calculate statistical significance, we utilized a custom R script that used a bootstrap method to account for the large sample sizes provided by ultra-deep sequencing as well as to correct for differences in sequencing sample size. All samples showed an increase in IGHV-D-J versus IGHM mutations after T cell activation. Five of 6 cases showed an increase in AID hostpot mutation frequency. AID coldspot mutation frequency decreased in 3/6 CLL cases. Percent transition mutation at C/G sites was higher than random in 2/6 CLL cases, which correlated with low frequencies of DNA polymerase eta hotspot mutation. In the other 4/6 CLL cases, the lower percent transitions at C/G sites may reflect the contribution of error-prone DNA repair. In summary, we developed a method to analyze ultra-deep IGHV-D-J sequences that revealed AID mutational characteristics in both U-CLL and M-CLL cells after activation with T-cell help in vitro or in vivo. These data are consistent with the hypothesis that AID, perhaps along with error-prone DNA repair, creates new mutations leading to the evolution of aggressive CLL. Disclosures: Rai: Sanofi: Membership on an entity’s Board of Directors or advisory committees; GSK: Membership on an entity’s Board of Directors or advisory committees; Teva: Membership on an entity’s Board of Directors or advisory committees; Genentech: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees.

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