ALBERT

Description: Abstract 3275 Poster Board III-1 Introduction: Leukemia stem cells (LSC) are rare, self-renewing cells capable of proliferation and differentiation into the bulk of cells that make up a leukemia. LSC, like normal tissue-specific stem cells, can be highly quiescent and resistant to apoptosis induced by drugs and radiotherapy that target rapidly dividing cells. While traditional chemotherapy may successfully eradicate the bulk of tumor cells, it often fails to kill LSC resulting in their reactivation and eventual relapse of disease. Apoptosis resistance in cancer often involves deregulation of Bcl-2 family proteins. Bcl-2 family expression also seems to have an important role in normal stem cell function as expression changes of Mcl-1, Bcl-2, and Bcl-XL in mouse hematopoietic stem and progenitors characterize the differentiation of hematopoietic cells to different lineages. Several studies have linked expression of Bcl-2 family members to the development of blast crisis chronic myeloid leukemia (BC CML) in vitro, however there has been little data on the role of Bcl-2 expression in apoptosis resistance in human LSC. Also, various data has shown that CML cells become increasingly resistant to BCR-ABL inhibition with progression to blast crisis. In BC CML, LSC are enriched in the progenitor population and can be serially transplanted in immunodeficient mice leading to BC CML in vivo. We hypothesize that human BC CML LSC may resist chemotherapy by overexpression of Bcl-2 family proteins and maintenance of quiescence. We analyzed Bcl-2 family protein expression in BC CML LSC and analyzed whether these cells were sensitive to chemotherapy treatment in vitro. We further analyzed whether BC CML LSC could maintain quiescence in vivo. Finally, we tested the efficacy of the broad spectrum Bcl-2 antagonist apogossypol on BC CML LSC in vitro and in vivo. Methods: Bcl-2 and Mcl-1 protein expression was measured in 1° BC CML LSC by intracellular FACS analysis and compared to expression in normal and chronic phase CML cells. CD34+ cells isolated from serially transplanted BC CML LSC were used for all subsequent studies. To assess whether BC CML cells maintained quiescence in vivo, they were stained with DiR, an infrared fluorescent cell membrane dye, transplanted into neonatal mice, and analyzed for DiR fluorescence 18 weeks later. Dividing cells will distribute DiR to all daughter cells leading to dilution of the dye and a decrease in fluorescent signal per cell. LSC drug resistance was tested in vitro by culturing the cells with etoposide, dasatinib, and apogossypol and by measuring apoptosis by FACS using annexin-V/7-AAD staining. Finally, apogossypol efficacy was tested in vivo in LSC transplanted mice. At 8 weeks post-transplantation, mice were treated for 3 weeks with apogossypol or vehicle and then analyzed for human hematopoietic cell engraftment and apoptosis by FACS. Results: 1° BC CML progenitors expressed significantly higher levels of Bcl-2 and Mcl-1 protein compared to normal cord blood and chronic phase CML cells. LSC cultured in vitro were also resistant to etoposide and dasatinib-induced apoptosis. Apogossypol treatment in vitro however led to a dose-dependent increase in cell death and apoptosis and resulted in a significant increase in the frequency of lin+ staining cells along with a significant shift in the frequency of the common myeloid progenitor (CMP) and granulocyte-macrophage progenitor (GMP) populations compared to vehicle treated controls. Mice transplanted with BC CML LSC developed diffuse myeloid sarcomas and had high levels of human engraftment in the liver, spleen, and bone marrow. Human cells in tumors and the liver were uniformly DiR- while cells engrafted in the spleen and bone marrow retained DiR fluorescence. In vivo treatment with apogossypol led to a significant reduction in human cell engraftment in mouse bone marrow compared to vehicle controls. Consistent with the results in vitro, there was also a significant increase in the frequency of lin+ staining cells in engrafted mouse spleens as well as a significant decrease in engrafted GMP in the mouse bone marrow. Conclusions: Our results demonstrate that BC CML LSC are highly resistant to conventional chemotherapy but are sensitive to apogossypol in vitro and in vivo. Broad-spectrum inhibition of Bcl-2 family proteins may help to eliminate CML LSC by inducing apoptosis as well as by inducing differentiation. Disclosures: Goff: Coronado Biosciences: Research Funding. Tesi:Coronado Biosciences: President and CEO. Jamieson:Coronado Biosciences: Research Funding. Jamieson:Coronado Biosciences: Research Funding.

Description: Abstract 1034 Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is a therapeutically recalcitrant malignancy that accounts for approximately 15% of pediatric and 25% of adult ALL cases. In leukemia, cancer stem cells constitute a relatively rare population of tumor cells that play a key role in cancer propagation and, like adult stem cells, have enhanced self-renewal potential. A previous report showed that following in vitro culture, CD34+/CD4- and CD34+/CD7- subfractions of T-ALL marrow were enriched for leukemia stem cells (LSC) capable of engrafting leukemia in nonobese diabetic/severe combined immune deficient mouse (NOD/SCID). However, difficulties in maintaining primary cultures of leukemia cells hampered investigations into the biology of T-ALL underscoring the need for a direct transplantation model to characterize human LSC in vivo and as a paradigm for screening candidate drugs that inhibit self-renewal pathways active in T-ALL. Experimental Procedures: Quantitative RT-PCR of NOTCH target gene expression and NOTCH mutation DNA sequencing analysis was performed on human CD34+ cells from T-ALL patient samples (n =12). To develop a humanized mouse model of T-ALL, CD34+ progenitors were lentivirally transduced with GFP-Luciferase Fusion protein (GLF) and transplanted intrahepatically into neonatal T, B, and NK cell deficient mice. In some experiments, FACS purified CD34+ subpopulations were transplanted at limiting dilution, including CD34+CD38+CD2+Lin- cells. Leukemic engraftment was monitored by in vivo bioluminescence imaging and analyzed by FACS detection of human CD34+ cells in liver, bone marrow, spleen and thymus when mice were sacrificed at 8–10 weeks post-transplant. NOTCH1 target gene expression was analyzed by q-RT-PCR in human CD34+ cells derived from engrafted tissues and NOTCH mutation analysis was performed by DNA sequencing on the same population. To assay LSC self-renewal, engrafted human CD34+ cells from bone marrow were transplanted into secondary and tertiary recipients. In serially transplanted mice, NOTCH1 target gene expression, NOTCH1 receptor expression was analyzed by FACS and NICD expression was assessed in the bone marrow by immunohistochemistry. Results: Q-RT-PCR data showed that NOTCH1, HES1 and c-MYC expression correlated with NOTCH 1 mutation status as well as the emergence of a CD34+CD2+Lin- population not evident in normal cord blood. We transplanted 12 T-ALL patient samples with detectable Notch1 expression and 100% of samples engrafted RAG 2-/- gamma c-/- mice. Transplanted LSC could be tracked for 10 weeks after transplant by in vivo bioluminescent imaging while Lin+ engraftment declined. Human CD34+/CD45+ cells, CD45+/CD34+/CD38+/Lin−/CD2+ cells were found in the bone marrow, thymus, spleen of the engrafted mice at 9–10 weeks post transplant or the end of dosing. Finally, human CD34+ cells engrafted secondary and tertiary recipients with T-ALL demonstrating their propensity for self-renewal and differentiation. Notch1 target gene and Hes1 expression was higher in patients with Notch1 mutation identified by sequencing. Conclusion: Serially transplantable candidate LSC retain high level NOTCH1 target gene expression and may be uniquely susceptible to targeted NOTCH1 receptor inhibition. Disclosures: Jamieson: Pfizer: Research Funding.

Description: Abstract 516 Introduction: Several studies have demonstrated the role of leukemia stem cells (LSC) in the development and maintenance of human chronic myeloid leukemia (CML). These cells, which first develop in chronic phase CML (CP CML) with acquisition of the BCR-ABL fusion protein, are often quiescent and can be highly resistant to apoptosis induced by drugs and radiotherapy that target rapidly dividing cells. Data has also shown that CML LSC become increasingly resistant to BCR-ABL inhibition with progression to blast crisis CML (BC CML). Bcl-2 family proteins are key regulators of apoptosis and have been shown by numerous studies to regulate cancer resistance to chemotherapy. This family of proteins has also been implicated in the development of BC CML, however most studies have focused on CML cell lines and their expression of Bcl-2 family proteins in vitro. Thus, there is relatively little data on expression of Bcl-2 family proteins in primary CML LSC and on the role of these proteins in regulating chemotherapy resistance in CML LSC in vivo. As Bcl-2 family proteins are known regulators of chemotherapy resistance we hypothesized that human BC CML LSC may overexpress these proteins compared to normal hematopoietic stem cells. We analyzed Bcl-2 family mRNA and protein expression in CP CML and BC CML LSC and compared this expression to normal cord blood stem and progenitor cells. We also analyzed whether these cells were sensitive to chemotherapy treatment in vitro. Finally, we tested whether a high potency pan-Bcl-2 inhibitor, 97C1, could effectively kill CML LSC in vitro and in vivo. Methods: Bcl-2 and Mcl-1 protein expression was measured in primary CP CML, BC CML, and normal cord blood cells using intracellular FACS. We also measured Bcl-2, Mcl-1, Bcl-X, and Bfl-1 mRNA expression in FACS sorted CD34+CD38+lin− cells (LSC) from these samples. For all drug studies we used either serially transplanted CD34+ cells derived from primary BC CML patient samples or primary CD34+ normal cord blood cells. In vitro drug responses were tested by culturing CD34+ cells either alone or in co-culture with a mouse bone marrow stromal cell line (SL/M2). Effects on colony formation and replating were also tested by culturing sorted CD34+CD38+lin− cells in methylcellulose in the presence and absence of drug. For in vivo testing of 97C1 we transplanted neonatal RAG2-/-yc-/- mice with CD34+ cells from 3 different BC CML and cord blood samples. Transplanted mice were screened for peripheral blood engraftment at 6–8 weeks post-transplant and engrafted mice were then treated for 2 weeks with 97C1 by IP injection. Following the treatment period the mice were sacrificed and hemotapoietic organs were analyzed for human engraftment by FACS. Results: BC CML progenitors expressed higher levels of Bcl-2 and Mcl-1 protein compared to normal cord blood and chronic phase CML cells. mRNA expression of Mcl-1, Bcl-X, and Bfl-1 was also increased in BC CML progenitors compared to CP CML progenitors. While BC CML LSC cultured in vitro were resistant to etoposide and dasatinib-induced cell death, 97C1 treatment led to a dose-dependent increase in cell death along with a dose-dependent decrease in the frequency of CD34+CD38+lin− cells compared to vehicle treated controls. While cord blood progenitor cells were also sensitive to 97C1 treatment they had an IC50 around 10 times higher than that for the BC CML cells (100nM versus 10nM). Importantly, 97C1 treatment did not inhibit cord blood colony formation or colony replating in vitro. Mice transplanted with BC CML LSC developed CML in 6–8 weeks post-transplant with diffuse myeloid sarcomas and engraftment of human CD34+CD38+lin− cells in the peripheral blood, liver, spleen, and bone marrow. In vivo treatment with 97C1 led to a significant reduction in both total human engraftment and engraftment of CD34+CD38+lin− cells in all hematopoietic organs analyzed. Conclusion: Our results demonstrate that BC CML LSC are resistant to conventional chemotherapy but are sensitive to 97C1 in vitro and in vivo. Broad-spectrum inhibition of Bcl-2 family proteins may help to eliminate CML LSC while sparing normal hematopoietic stem and progenitor cells. Disclosures: Jamieson: CoronadoBiosciences: Research Funding; CIRM: Research Funding.

Description: Abstract 2735 Leukemia stem cells (LSC) play a crucial role in the development and progression of chronic myeloid leukemia (CML). Although BCR-ABL targeted tyrosine kinase inhibitors (TKI), such as dasatinib, can eradicate the majority of CML cells, they frequently fail to eliminate the dormant, niche-resident LSC that are hypothesized to drive CML relapse. Cumulative evidence from CML cell lines and CD34+ primary patient cells suggests that increased expression of pro-survival BCL2 family members contributes to TKI resistance and CML progression. However there is a relative dearth of data on BCL2 family expression in primary CML LSC and on the role of these proteins in TKI resistance in selective niches. Full transcriptome RNA sequencing revealed that LSC switch from pro-apoptotic to pro-survival BCL2 family member splice isoform expression during progression from chronic phase to blast crisis CML. Using splice isoform-specific qRT-PCR, we identified overrepresentation of long (pro-survival) compared with short (pro-apoptotic) MCL1, BCLX, and BCL2 isoforms in blast crisis LSC compared with chronic phase and normal progenitors. Following intrahepatic transplantation of blast crisis LSC into neonatal RAG2−/−gc−/− mice, LSC engrafted in the marrow niche were quiescent, were dasatinib resistant and upregulated BCL2 expression. These data led us to speculate that inhibition of BCL2 in dasatinib-resistant LSC may sensitize LSC to TKI therapy. Treatment with a high-potency, novel pan-BCL2 family inhibitor, sabutoclax, in vitro led to a dose-dependent increase in apoptosis along with a decrease in the frequency of leukemic progenitors compared to vehicle treated controls. Normal human cord blood progenitor cells were less sensitive to sabutoclax treatment with IC50 approximately five times higher than that for blast crisis CML cells (210 nM versus 43 nM). Moreover, sabutoclax treatment did not inhibit cord blood colony formation or colony replating in vitro. Treatment of CML LSC-transplanted mice with sabutoclax led to a significant reduction in LSC burden in all hematopoietic organs analyzed. Sabutoclax treatment in vivo also sensitized surviving bone marrow blast crisis LSC to dasatinib treatment ex vivo. Importantly, there was no reduction in normal progenitor engraftment in bone marrow following sabutoclax treatment. These results demonstrate that marrow niche blast crisis CML LSC survival is driven by overexpression of multiple pro-survival BCL2 family isoforms rendering them susceptible to a novel pan, BCL2 antagonist, sabutoclax, at doses that spare normal hematopoietic progenitors. While BCL2 splice isform switching promotes LSC survival and TKI resistance, pan-BCL2 family member inhibition with sabutoclax eliminates LSC and may form the cornerstone of a clinical strategy to avert cancer progression and relapse. Disclosures: No relevant conflicts of interest to declare.

Description: Many cancers evade immune rejection by suppressing major histocompatibility class I (MHC-I) antigen processing and presentation (AgPP). Such cancers do not respond to immune checkpoint inhibitor therapies (ICIT) such as PD-1/PD-L1 [PD-(L)1] blockade. Certain chemotherapeutic drugs augment tumor control by PD-(L)1 inhibitors through potentiation of T-cell priming but whether and how chemotherapy enhances MHC-I–dependent cancer cell recognition by cytotoxic T cells (CTLs) is not entirely clear. We now show that the lysine acetyl transferases p300/CREB binding protein (CBP) control MHC-I AgPPM expression and neoantigen amounts in human cancers. Moreover, we found that two distinct DNA damaging drugs, the platinoid oxaliplatin and the topoisomerase inhibitor mitoxantrone, strongly up-regulate MHC-I AgPP in a manner dependent on activation of nuclear factor kappa B (NF-κB), p300/CBP, and other transcription factors, but independently of autocrine IFNγ signaling. Accordingly, NF-κB and p300 ablations prevent chemotherapy-induced MHC-I AgPP and abrogate rejection of low MHC-I–expressing tumors by reinvigorated CD8+ CTLs. Drugs like oxaliplatin and mitoxantrone may be used to overcome resistance to PD-(L)1 inhibitors in tumors that had “epigenetically down-regulated,” but had not permanently lost MHC-I AgPP activity.

Description: Despite the development of next-generation antiandrogens, metastatic castration-resistant prostate cancer (mCRPC) remains incurable. Here, we describe a unique semisynthetic bispecific antibody that uses site-specific unnatural amino acid conjugation to combine the potency of a T cell–recruiting anti-CD3 antibody with the specificity of an imaging ligand (DUPA) for prostate-specific membrane antigen. This format enabled optimization of structure and function to produce a candidate (CCW702) with specific, potent in vitro cytotoxicity and improved stability compared with a bispecific single-chain variable fragment format. In vivo, CCW702 eliminated C4-2 xenografts with as few as three weekly subcutaneous doses and prevented growth of PCSD1 patient-derived xenograft tumors in mice. In cynomolgus monkeys, CCW702 was well tolerated up to 34.1 mg/kg per dose, with near-complete subcutaneous bioavailability and a PK profile supporting testing of a weekly dosing regimen in patients. CCW702 is being evaluated in a first in-human clinical trial for men with mCRPC who had progressed on prior therapies (NCT04077021).