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Purpose:The Polo-like kinase 1 (PLK1) is a master regulator of mitosis and overexpressed in acute myeloid leukemia (AML). We conducted a phase Ib study of the PLK1 inhibitor, onvansertib, in combination with either low-dose cytarabine (LDAC) or decitabine in patients with relapsed or refractory (R/R) AML.Patients and Methods:Onvansertib was administered orally, in escalating doses, on days 1–5 in combination with either LDAC (20 mg/m2; days 1–10) or decitabine (20 mg/m2; days 1–5) in a 28-day cycle. The primary endpoint was to evaluate first-cycle dose-limiting toxicities and the MTD. Secondary and exploratory endpoints included safety, pharmacokinetics, antileukemic activity, and response biomarkers.Results:Forty patients were treated with onvansertib (12–90 mg/m2) in combination with LDAC (n = 17) or decitabine (n = 23). Onvansertib was well tolerated with most grades 3 and 4 adverse events related to myelosuppression. In the decitabine arm, the MTD was established at 60 mg/m2, and 5 (24%) of the 21 evaluable patients achieved complete remission with or without hematologic count recovery. Decrease in mutant circulating tumor DNA (ctDNA) during the first cycle of therapy was associated with clinical response. Engagement of the PLK1 target, TCTP, was measured in circulating blasts and was associated with greater decrease in bone marrow blasts.Conclusions:The onvansertib and decitabine combination was well tolerated and had antileukemic activity particularly in patients with target engagement and decreased mutant ctDNA following treatment. This combination will be further investigated in the ongoing phase II trial.

Suppl Figures 1-3, Suppl Table 1 Supplemental Figure 1. Study schema. Supplemental Figure 2. Overall survival in patients treated with ENTO + 7+3. OS is censored on the date the patient was last known to be alive (if it was not known the patient had died by the end of study follow-up). Supplemental Figure 3. Heat map showing single patients (each column) by HOXA9 and MEIS1 categorization, cytogenetic or molecular risk and outcomes.

Detailed list of variants identified by targeted-NGS

Purpose:Spleen tyrosine kinase (SYK) signaling is a proposed target in acute myeloid leukemia (AML). Sensitivity to SYK inhibition has been linked to HOXA9 and MEIS1 overexpression in preclinical studies. This trial evaluated the safety and efficacy of entospletinib, a selective inhibitor of SYK, in combination with chemotherapy in untreated AML.Patients and Methods:This was an international multicenter phase Ib/II study, entospletinib dose escalation (standard 3+3 design between 200 and 400 mg twice daily) + 7+3 (cytarabine + daunorubicin) in phase Ib and entospletinib dose expansion (400 mg twice daily) + 7+3 in phase II.Results:Fifty-three patients (n = 12, phase Ib and n = 41, phase II) with previously untreated de novo (n = 39) or secondary (n = 14) AML were enrolled (58% male; median age, 60 years) in this study. The composite complete response with entospletinib + 7+3 was 70%. Patients with baseline HOXA9 and MEIS1 expression higher than the median had improved overall survival compared with patients with below median HOXA9 and MEIS1 expression. Common adverse events were cytopenias, febrile neutropenia, and infection. There were no dose-limiting toxicities. Entospletinib-related skin rash and hyperbilirubinemia were also observed.Conclusions:Entospletinib with intensive chemotherapy was well-tolerated in patients with AML. Improved survival was observed in patients with HOXA9/MEIS1 overexpression, contrasting published data demonstrating poor survival in such patients. A randomized study will be necessary to determine whether entospletinib was a mediator this observation.

Purpose: The Polo-like kinase 1 (PLK1) is a master regulator of mitosis and overexpressed in acute myeloid leukemia (AML). We conducted a phase Ib study of the PLK1 inhibitor, onvansertib, in combination with either low-dose cytarabine (LDAC) or decitabine in patients with relapsed or refractory (R/R) AML. Patients and Methods: Onvansertib was administered orally, in escalating doses, on days 1–5 in combination with either LDAC (20 mg/m2; days 1–10) or decitabine (20 mg/m2; days 1–5) in a 28-day cycle. The primary endpoint was to evaluate first-cycle dose-limiting toxicities and the MTD. Secondary and exploratory endpoints included safety, pharmacokinetics, antileukemic activity, and response biomarkers. Results: Forty patients were treated with onvansertib (12–90 mg/m2) in combination with LDAC (n = 17) or decitabine (n = 23). Onvansertib was well tolerated with most grades 3 and 4 adverse events related to myelosuppression. In the decitabine arm, the MTD was established at 60 mg/m2, and 5 (24%) of the 21 evaluable patients achieved complete remission with or without hematologic count recovery. Decrease in mutant circulating tumor DNA (ctDNA) during the first cycle of therapy was associated with clinical response. Engagement of the PLK1 target, TCTP, was measured in circulating blasts and was associated with greater decrease in bone marrow blasts. Conclusions: The onvansertib and decitabine combination was well tolerated and had antileukemic activity particularly in patients with target engagement and decreased mutant ctDNA following treatment. This combination will be further investigated in the ongoing phase II trial.

Purpose: Spleen tyrosine kinase (SYK) signaling is a proposed target in acute myeloid leukemia (AML). Sensitivity to SYK inhibition has been linked to HOXA9 and MEIS1 overexpression in preclinical studies. This trial evaluated the safety and efficacy of entospletinib, a selective inhibitor of SYK, in combination with chemotherapy in untreated AML. Patients and Methods: This was an international multicenter phase Ib/II study, entospletinib dose escalation (standard 3+3 design between 200 and 400 mg twice daily) + 7+3 (cytarabine + daunorubicin) in phase Ib and entospletinib dose expansion (400 mg twice daily) + 7+3 in phase II. Results: Fifty-three patients (n = 12, phase Ib and n = 41, phase II) with previously untreated de novo (n = 39) or secondary (n = 14) AML were enrolled (58% male; median age, 60 years) in this study. The composite complete response with entospletinib + 7+3 was 70%. Patients with baseline HOXA9 and MEIS1 expression higher than the median had improved overall survival compared with patients with below median HOXA9 and MEIS1 expression. Common adverse events were cytopenias, febrile neutropenia, and infection. There were no dose-limiting toxicities. Entospletinib-related skin rash and hyperbilirubinemia were also observed. Conclusions: Entospletinib with intensive chemotherapy was well-tolerated in patients with AML. Improved survival was observed in patients with HOXA9/MEIS1 overexpression, contrasting published data demonstrating poor survival in such patients. A randomized study will be necessary to determine whether entospletinib was a mediator this observation.

Background The SARS–CoV-2 pandemic has assaulted all aspects of daily life. Medical professionals in oncology face additional challenges with balancing prompt cancer diagnosis and urgent treatment against potential COVID-19 exposure risk in these high-risk patients. We designed this prospective freewill study to offer testing for SAR2-CoV-2 viral RNA and/or anti-COVID-19, respectively in asymptomatic medical and research staff who work in direct contact with cancer patients. The overall goal was to evaluate the prevalence of infection in this group of asymptomatic healthcare providers to reduce exposure of cancer patients to asymptomatic staff. Methods Asymptomatic medical and research staff who work in direct contact with cancer patients were asked to voluntarily be tested for either SARS–CoV-2 viral RNA or antibodies or both. Either NP swabs and/or blood samples (EDTA tube) were collected. Tests are performed at Sinochips Kansas LLC, Sinochips Diagnostics (CLIA number:17D2176068, CAP number: 8709463). The PCR test is performed with FDA authorized 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel EUA. The Elecsys® Anti-SARS-CoV-2 (Roche Diagnostics) immunoassay was used to qualitative detection of antibodies to SARS-CoV-2 in human plasma. Results From 06/18/2020 to 12/18/2020, 861 participated in the study. 1095 tests were completed for SAR2-CoV-2 virus infection, and 918 were completed for antibody. Amount participants, 530 had both virus and antibody tested. 235 were tested more than once for viral infection and 166 were tested more than once for the antibody. Median age of participants was 39 years (IQR 32-51 years). Among these 84.7% were females, 84.4% white, 6.7% African American, 4.8% Asian and 84.7% non-Hispanic. The cumulative incidence of a positive test for the virus was 2.2% (16/712), and for the antibody test was 3.8% (26/679). 5 had both viral and antibody tests positive, with an average time of 4.1 weeks from viral testing positivity to detectable antibody among 3 cases and 2 cases with both viral infection and antibody detected at same time. There were 3 cases virus was detected more than once after turning positive. 2 remained positive at 16 and 22 days after initial test and one turned negative at 36 days as of last follow up. There were 7 cases where the antibody was tested more than once after turning positive and all 7 remained positive as of last follow up (range 7-103 days). Conclusion Prospective voluntary testing in asymptomatic medical and research staff who work in direct contact with cancer patients was feasible and resulted in identification of asymptomatic carriers who then placed in quarantine, thereby limiting exposure to cancer patients. Medical and research staff who work with cancer patients are general very cautious and the frequency of infections were significantly lower than general society. In addition, it seems that 1) virus and antibody may co-exist in the same person after exposure, and 2) the antibody may last for a relatively long time. Citation Format: Weijing Sun, Andrew K. Godwin, Kathan Mehta, Natalie Streeter, Elizabeth Wulff-Burchfield, Anup Kasi, Tara L. Lin, Joaquina Baranda, Joseph McGuirk, Ziyan Pessetto, Adam Pessetto, B.J. Broome, Terry Tsue, Roy Jensen. Prospective voluntary SARS-CoV-2 virus and anti-COVID-19 antibody tests in asymptomatic medical and research staff who work in direct contact with cancer patients: A single center study [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2021 Feb 3-5. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(6_Suppl):Abstract nr P13.

Background: Chemotherapy-resistant leukemia stem cells (LSC) are responsible for disease relapse in many patients with Acute Myeloid Leukemia (AML). The Wnt/β-catenin and PI3K/Akt pathways are frequently activated in cancer and interact to stimulate stem cell expansion through phosphorylation of β-catenin by Akt (pS552-β-catenin). We hypothesize that targeting pS552-β-catenin may inhibit chemoresistant LSCs. High-throughput screening identified that anthracyclines doxorubicin (DXR) and daunorubicin (DNR) inhibit Akt:β-catenin interaction at significantly lower doses than conventionally used. Using a mouse leukemia model, we repurposed DXR as a targeted therapy for pS552-β-catenin inhibition rather than a broadly cytotoxic agent. In contrast to the typical dose, which may stimulate expansion of resistant LSC, targeted/low-dose DXR inhibited chemoresistant LSC expansion and significantly reduced LSC tumorigenic potential. AML patients typically receive high-dose DNR but often relapse due to persistence of therapy-resistant LSCs, which frequently express pS552-β-catenin. Here, we use a metronomic approach to test the ability of low-dose anthracycline therapy to target therapy-resistant LSCs. Methods: A proof of concept trial (NCT02914977) was designed to measure the ability of low dose DNR to inhibit pS552-β-catenin in LSCs of adult relapsed/refractory AML patients. A protocol amendment added a second cohort of patients with newly diagnosed AML undergoing standard chemotherapy with DNR and cytarabine (7+3 induction). Treatment in each cohort consisted of DNR given at a dose of 6.75mg/m2/day x 5 days. Patients with relapsed/refractory AML had bone marrow aspiration pre-treatment prior to day 1, followed by DNR on days 1-5, and a post-treatment bone marrow aspiration on day 8. Patients with newly diagnosed AML were treated with conventional 7+3 chemotherapy. A sample from the day 14 bone marrow was collected. DNR was given on days 15-19. Bone marrow aspiration was performed with blood count recovery and samples sent for LSC marker assessment. In this study, we will determine the safety, pharmacokinetics, and pharmacodynamic effects of low dose DNR. The LSC population and phosphorylation status of β-catenin will be measured pre- and post-treatment. Two or more prior induction attempts are required for study entry for refractory patients; relapsed patients require only one prior induction. The trial has enrolled 14 patients: 11 in the relapsed cohort and 3 in the newly diagnosed cohort. A total of 18 patients are planned. In this pilot proof of concept trial, we will demonstrate the feasibility of serial bone marrow aspiration to measure biomarkers of LSC response. This clinical proof of concept trial design with serial bone marrow aspirations may also be used to rapidly assess the validity of other LSC markers and targeted agents. Citation Format: Tara L. Lin, John M. Perry, Xi He, Gregory Reed, Na Zhang, Scott Weir, Joseph McGuirk, Linheng Li. Low-dose daunorubicin to target leukemia stem cells in newly diagnosed and relapsed/refractory AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT103.

Background: Despite recent advances in the understanding of the biology and development of new agents for patients with acute myeloid leukemia (AML), prognosis remains poor with most patients (pts) succumbing to their disease. PLK1 is a serine/threonine kinase, a master regulator of cell-cycle progression, and is overexpressed in numerous cancer types including AML. Onvansertib is a third generation, orally active and highly selective PLK1 inhibitor with a ~24-hour half-life that demonstrates activity in preclinical AML models both as a single agent and in combination with low-dose cytarabine (LDAC). A Phase I study showed that Onvansertib was well tolerated in pts with solid tumors. Methods: The goal of this Phase Ib (NCT03303339) study is to test the safety of Onvansertib in combination with either LDAC or Decitabine in relapsed or refractory AML. Efficacy, pharmacokinetics and pharmacodynamics of the combination are secondary endpoints. Pts are treated with Onvasertib for 5 days in combination with either LDAC (20 mg/m2 SC qd x 10d) or Decitabine (20 mg/m2 IV qd x 5d) over a flexible 21 to 28-day cycle, with the next cycle initiated based on recovery of cell counts. Each arm follows a standard dose escalation design with 50% increments in successive cohorts of 3 pts. Dose limiting toxicity (DLT) is evaluated during the 1stcycle. As of January 2, 2019, 21 pts have enrolled. The starting dose of Onvansertib was 12mg/m2 and was escalated to 18, 27 and 40mg/m2 in subsequent cohorts. In both arms, no treatment-related serious adverse events and deaths or DLTs occurred at the first 3 doses and treatment at 40mg/m2 is ongoing. Additional objectives include pharmacokinetics, preliminary anti-leukemic activity and correlative biomarker and pharmacodynamics analyses. Assessment of PLK1 inhibition is being determined in pts by changes in phosphorylation status of the translationally controlled tumor protein (TCTP), which is a PLK1 substrate, and is being evaluated not only in terms of response to treatment, but also as a potentially identifiable marker ex vivo for pts that may be more responsive to therapy. Genomic and gene expression analysis are assessed to identify biomarkers that may also be associated with response to treatment. Citation Format: Amer M. Zeidan, Pamela Becker, Alexander I. Spira, Prapti A. Patel, Gary J. Schiller, Michaela L. Tsai, Tara L. Lin, Maya Ridinger, Mark Erlander, Sandra L. Silberman, Jorge E. Cortes. Phase Ib safety, preliminary anti-leukemic activity and biomarker analysis of the polo-like kinase 1 (PLK1) inhibitor, onvansertib, in combination with low-dose cytarabine or decitabine in patients with relapsed or refractory acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT102.

Patients with acute leukemia harbor chemotherapy-resistant leukemia stem cells (LSC) which ultimately lead to disease relapse in many patients. Clinical trial design of LSC targeted therapy is a challenge. The majority of leukemia cells are differentiated leukemia cells, with LSC comprising Citation Format: Tara L. Lin, John M. Perry, Xi He, Gregory Reed, Na Zhang, Scott Weir, Joseph P. McGuirk, Linheng Li. Biomarkers of response to leukemia stem cell targeted therapy with low dose daunorubicin in relapsed/refractory acute leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT137.

Introduction: Targeted therapy for KMT2A (mixed lineage leukemia [MLL]) rearranged acute leukemia (AL) is lacking. KMT2A regulates leukemic stem cell transcription factors HOXA9 and MEIS1. Spleen tyrosine kinase (SYK) signaling induces MEIS1 in conjunction with HOXA9. We hypothesize that this regulatory loop may be sensitive to SYK inhibition with Entospletinib (ENTO) a highly selective, oral SYK inhibitor. In this analysis, we evaluate the efficacy of ENTO in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) with t(v;11q23.3) KMT2A/MLL gene rearrangements. Methods: Patients with KMT2A rearranged AL in two different clinical trials were included. AML patients (NCT02343939) received ENTO 400mg BID monotherapy for up to 14 days prior to and with induction chemotherapy (cytarabine 100 mg/m2 for 7 days plus daunorubicin 60 mg/m2 for 3 days) if previously untreated, or ENTO monotherapy alone if relapsed/refractory (R/R). R/R B-ALL patients received ENTO monotherapy for 7 days prior to and with vincristine and dexamethasone in a phase 1 study (NCT02404220). Results: 18 patients with KMT2A rearranged AL were treated. Untreated AML (n=10) patients had a median age of 49 years. One patient was in a morphologic leukemia-free state after ENTO monotherapy (before chemotherapy). After induction chemotherapy plus ENTO, seven patients had morphologic (CR) and cytogenetic (CRc) remission, but two of them had incomplete count recovery (CRi). The composite CR rate (CR/CRi/CRc) was 90%: 5 CRc, 3 CRi (CR/CRc with incomplete count recovery), and 1 CR. Six patients underwent allogeneic stem cell transplantation (SCT) in CR1. After median follow-up of 9.9 months one patient who achieved CR relapsed, with persistence of t (6;11) and an NRAS mutation. Median overall survival and event-free survival have not been reached. In R/R AML (n=6), patients had a median age of 48 years, with 2 median prior therapies. One had CR and one had CRi (but normal cytogenetics) and both received SCT. In all, 3 AML patients (1 newly diagnosed, 2 R/R) responded to ENTO monotherapy alone. In R/R ALL (n=2), median age was 59 years. Both patients had t (4;11) along with other cytogenetic abnormalities with 2 prior therapies. Both patients achieved CR with loss of KMT2A on cytogenetic testing, and one patient received SCT. Overall, ENTO was safe and well tolerated, even in combination with chemotherapy. Conclusions: KMT2A rearranged AL is sensitive to ENTO with CR observed on monotherapy in AML and high response rates in AL patients treated with combination therapy. This represents the first documentation of CR with small molecule monotherapy (n=3) in this genetic subgroup, which typically portends a poor prognosis. Correlative biomarker studies evaluating HOXA9/MEIS1 in patients with t(v;11q23.3) KMT2A rearranged leukemia treated with ENTO are pending. Citation Format: Alison R. Walker, John C. Byrd, William Blum, Tara Lin, Howland E. Crosswell, Danjie Zhang, Jie Gao, Arati V. Rao, Mark D. Minden, Wendy Stock. High response rates with entospletinib in patients with t(v;11q23.3);KMT2A rearranged acute myeloid leukemia and acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 819.

Objectives: Acute myeloid leukemia (AML) is a heterogeneous disease with diverse genetic abnormalities present in all ages, but mainly prevalent in an elderly population with an average age of above 60 years. The treatment of elderly AML remains a formidable challenge as the long-term outcomes of elderly AML patients have not improved in the last three decades, with ≤5% overall survival recorded at 5 years, calling for novel treatment options. Epigenetic therapy has a significant impact on the management of hematologic malignancies. Recent findings show that using very low dosages of decitabine depletes DNA methyl transferase 1 (DNMT1) without cytotoxicity. Decitabine-mediated epigenetic therapy does not induce FLT3 (fms-like tyrosine kinase 3) ligand, which hinders the effectiveness of FLT3 inhibitors. The inhibition of FLT3 and DNMT1 is associated with terminal myeloid differentiation of AML cells. Therefore, combining the inhibitors of FLT3 and DNMT1 may be an effective therapeutic approach for the treatment of poor-risk AML. Ponatinib is a third-generation receptor tyrosine kinase inhibitor. Various in vitro and in vivo preclinical studies demonstrated antileukemic activities of ponatinib against AML cells bearing FLT3-ITD mutations. Methods: Apoptosis and CD11b were measured by flow cytometry. FLT3 signaling and DNMT1 levels were analyzed by immunoblotting. Results: FLT3-ITD expressing AML cell lines MV4-11, MOLM-13, and MOLM-14 were used in the study. Co-treatment with decitabine (10-100 nM) and ponatinib (2 nM) in AML cells induced apoptosis in association with PARP cleavage. Increased levels of pro-apoptotic protein BAD and decreased levels of anti-apoptotic protein MCL1 were observed in these treated cells. The combination of decitabine and ponatinib also showed similar effects in primary AML cells expressing FLT3-ITD. The Western blot analysis demonstrated that treatment of decitabine decreased levels of DNMT1 and ponatinib inhibited FLT3 signaling and activation of downstream effectors STAT5, AKT, and ERK1/2 with induction of PU.1 a key regulator of myeloid differentiation. Co-treatment with ponatinib and decitabine using low concentrations in MV4-11 and MOLM14 cells induced myeloid differentiation. The percentage of differentiated cells was measured by increased surface expression of CD11b analyzed by flow cytometry and granulocytic/monocytic morphology examined by Wright-Giemsa staining. Conclusions: Mechanistically, the hypomethylating agent initiates the differentiation process and FLT3 inhibition augments differentiation leading to apoptosis of AML cells. Altogether, these preclinical findings of downregulation of DNMT1 and induction of PU.1 are a novel differentiation approach to induce apoptosis in AML cells and warrant future therapeutic potential for the treatment of AML patients expressing FLT3 mutations. Citation Format: Sudhakiranmayi Kuravi, Myles Taylor, Tara L. Lin, Jensen Roy, Joseph McGuirk, Ramesh Balusu. A targeted differentiation therapy for the treatment of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4821.

Misfolded or aggregated proteins have two fates: they are either refolded with the help of chaperones or degraded by the proteasome. Cells also have an alternative pathway that involves intracellular “storage bins” for misfolded intracellular proteins known as aggresomes. Aggresomes recruit motor proteins that transport misfolded or aggregated proteins to chaperones and proteasomes for subsequent destruction. There is emerging evidence that inhibiting the aggresome pathway leads to accumulation of misfolded proteins and apoptosis in tumor cells through autophagy. We discuss the role of aggresomes in cancer and the potential to target this pathway for therapy. [Cancer Res 2008;68(8):2557–60]

Introduction: Multiple Myeloma (MM) is an incurable cancer with a pre-malignant clonal phase of disease called monoclonal gammopathy of undetermined significance (MGUS). Most patients with MGUS do not develop MM and the biology underlying this potential transformation is unclear. Investigations to prevent the development of MM from MGUS is limited by infrequent MGUS progression. MGUS cells have historically proven difficult to grow in vitro because of slow rates of proliferation and difficulty in sustaining cell cultures. An in vitro model of MGUS that circumvents these challenges by recreating a supportive bone marrow microenvironment will allow further investigation into the pathogenesis of disease progression. Herein, we examine a novel stroma-based in vitro model of MGUS and MM using bone marrow stromal cells to support and maintain a bi-directional relationship within the clonal plasma cell population. Aim: To grow MGUS derived plasma cells by recreating a supportive bone marrow microenvironment using a 3-dimensional in vitro model. Methods: We collected a CD38+ cell fraction and a bone marrow stromal fraction from patients with plasma cell dyscrasias using a Miltenyl Biotec column Separator. We seeded stromal cells from a female, kappa expressing MGUS patient or a healthy donor onto a polyglycolic acid/ poly L-lactic acid 90/10 (PLGA) copolymer scaffold. Then, a CD38+ cell fraction from a male patient with lambda secreting PCL or MM cell line U266 or MM1.s (both male, lambda expressing cell lines) were added to create 3D co-culture conditions. For comparison, a 2 dimensional co-culture condition was created by growing MM patient cells or cell lines over a bone marrow stromal cell layer without scaffold.. After one week, samples were formalin fixed and sectioned for immunohistochemistry (IHC) and stained with CD138, kappa, lambda, and ki-67. Fluorescent in situ hybridization (FISH) was used to label X and Y chromosomes. Results: IHC demonstrated proliferation of CD138+ cells evident by Ki-67 expression in in 3-D co-culture systems. There was no growth of the patient derived CD38+ cells in 2-D co-culture and the proliferation of of cell lines was diminished in 2-D versus 3-D. When seeded on healthy donor stroma in 3-D, the CD38+ cells from all sources were lambda restricted. When seeded on the MGUS stroma, the MM1.s cells were lambda restricted while PCL cells showed both kappa and lambda expression with a dominance of kappa expression, and U266 cells showed kappa restriction. FISH demonstrated a female chromosomal pattern with X,Y in the plasma cells from the 3D co-cultures in the case of U266 or PCL cell 3-D co-culture with MGUS stroma. Conclusion: This is the first demonstration of in vitro growth of plasma cells from a patient with MGUS. We speculate the aggressive plasma cells from the U266 cell line and the PCL sample induced the growth of a CD38- cell fraction and caused differentiation to plasma cells. Citation Format: Brea C. Lipe, Omar Aljitawi, Tara Lin, Joseph Fontes. Growth of monoclonal gammopathy of undetermined significance (MGUS) in a 3-dimensional co-culture in vitro model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4026. doi:10.1158/1538-7445.AM2015-4026

Background: Aberrant HH pathway signaling has been implicated in human malignancies ranging from semi-malignant tumors of the skin to highly aggressive cancers of the brain, lung, pancreas, breast, prostate, and lymphohematopoietic lineages. Dysregulation of this pathway contributes to uncontrolled proliferation, invasion, metastasis, evasion of apoptosis, and resistance to chemotherapy. Results: BMS-833923 is a potent inhibitor of SMO, a GPCR-like 7-transmembrane receptor that is a critical regulator of the HH pathway. In vitro, BMS-833923 inhibits the expression of downstream effectors in the HH pathway (GLI1 and PTCH1) in cell lines that express wild-type SMO and those which express activated mutant forms of SMO (IC50 values of 6–35 nM). In FACS-based binding assays, BMS-833923 inhibits BODIPY cyclopamine binding to SMO in a dose-dependent manner with an IC50 of 21 nM. Prior work has demonstrated that HH pathway blockade inhibits the in vitro clonal expansion of multiple myeloma (MM) precursor cells in both established cell lines and in clinical samples from patients with MM (Peacock et al 2007, PNAS 104:4048). Consistent with these observations, BMS-833923 inhibited the in vitro growth of MM clones and proportion of ALDH+ cancer stem cells derived from bone marrow samples from subjects with MM. Furthermore, BMS-833923 inhibited the clonogenic growth of multiple tumor cell lines derived from patients with hematological malignancies including CML, ALL and AML, suggesting that targeting the HH pathway may have broad therapeutic utility for patients with hematologic malignancies. In vivo pharmacodynamic studies show that BMS-833923 robustly inhibits HH pathway activity with a long duration of action after a single oral dose in medulloblastoma and pancreatic carcinoma xenograft models. The pharmacodynamic effects of BMS-833923 observed in these models translate into tumor growth inhibition at well-tolerated doses. These results are consistent with pharmacokinetic parameters demonstrated in nonclinical studies. Conclusions: BMS-833923 is an orally bioavailable, potent and selective inhibitor of the HH pathway as measured by in vitro cell-based assays and in vivo pharmacodynamic and efficacy models, supporting the clinical development of BMS-833923 in both hematological and non-hematological malignancies. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B192.