ALBERT

Description: Cu 2 ZnSn(S,Se) 4 (CZTSSe) solar cells typically exhibit high short-circuit current density ( J sc ), but have reduced cell efficiencies relative to other thin film technologies due to a deficit in the open-circuit voltage ( V oc ) , which prevent these devices from becoming commercially competitive. Recent research has attributed the low V oc in CZTSSe devices to small scale disorder that creates band tail states within the absorber band gap, but the physical processes responsible for this V oc reduction have not been elucidated. In this paper, we show that carrier recombination through non-mobile band tail states has a strong voltage dependence and is a significant performance-limiting factor, and including these effects in simulation allows us to simultaneously explain the V oc deficit, reduced fill factor, and voltage-dependent quantum efficiency with a self-consistent set of material parameters. Comparisons of numerical simulations to measured data show that reasonable values for the band tail parameters (characteristic energy, capture rate) can account for the observed low V oc , high J sc , and voltage dependent collection efficiency. These results provide additional evidence that the presence of band tail states accounts for the low efficiencies of CZTSSe solar cells and further demonstrates that recombination through non-mobile band tail states is the dominant efficiency limiting mechanism.

Description: Duvelisib (IPI-145), a dual inhibitor of phosphoinositide 3-kinase (PI3K)-δ and -γ, has shown clinical activity in treatment-naïve and relapsed/refractory chronic lymphocytic leukemia (CLL) patients. Clinically, duvelisib results in a redistribution of malignant B cells and concomitant reduction in nodal enlargement. These effects are believed to be due to important roles of PI3K- δ and -γ in CXCL12-mediated CLL cell migration (Peluso 2014), cytokine-induced CLL B-cell proliferation, and BCR-stimulated B-cell survival (Balakrishnan 2015). Additional data suggest an effect of duvelisib on the tumor supporting cells of the CLL microenvironment. This includes preclinical studies demonstrating that PI3K-γ inhibition blocks normal T cell migration toward tumor chemokines and prevents murine bone marrow-derived M2 macrophage polarization (Peluso 2014), as well as clinical data in CLL patients receiving duvelisib showing reduced serum levels of myeloid and T cell-secreted cytokines and chemokines (Douglas 2015). To further characterize duvelisib's effect on CLL cells and the tumor microenvironment (TME), a murine xenograft model using primary human CLL cells was employed. We first studied duvelisib's effect on CLL B- and T-cell migration in vivo. CLL PBMCs (n=2; 1 IGHV unmutated (U)-CLL, 1 IGHV mutated (M)-CLL) pre-treated with duvelisib for 48 hours were injected retro-orbitally into NOD-scid IL2Rgammanull (NSG) mice. B- and T-cell localization in tissues and circulation was studied 1 and 24 hours post-injection. Duvelisib treatment (1000 nM) prevented the egress of CLL B and T cells from the circulation into the spleen, indicating impaired homing of CLL B and T cells. To better define the effect of duvelisib on T-cell migration, T cells from CLL patients (n=3; 2 U-CLL, 1 M-CLL) treated ex vivo with duvelisib at 1, 10, 100 and 1000 nM were injected into mice and analyzed for their trafficking 24 hours later. Inhibition of T-cell homing to spleen was dose dependent, with only 100 and 1000 nM having significant effects. Given duvelisib's cellular IC50s for PI3K isoforms, these results suggest that impaired T-cell migration is due to PI3K-γ inhibition, and studies with isoform-selective PI3K-δ and PI3K-γ inhibitors are currently underway to examine this possibility. The effect of duvelisib on CLL T-cell proliferation was evaluated after in vitro activation with anti-CD3/28 Dynabeads plus IL2 (n=6; 3 U-CLL, 3M-CLL). In duvelisib treated cells, CD4+, but not CD8+, T-cell proliferation was inhibited at doses of 100 and 1000 nM, suggesting a role for PI3K-γ. The effects of duvelisib on CLL B- and T-cell growth in vivo (n=4; 2 U-CLL, 2 M-CLL) were then studied. Autologous CLL T cells were stimulated as above and injected with CLL PBMCs into NSG mice. Animals treated orally with duvelisib for 3 weeks at 100 mg/kg/day had preferentially reduced CD4+ T-cell recovery from spleens, thereby decreasing the CD4 to CD8 ratio. In each case, duvelisib treatment reduced the number of splenic CLL B cells. This reduction reflected inhibition of both CLL cell proliferation and survival, since duvelisib treatment decreased the percentage of cycling CLL cells and increased the percentage of apoptotic B cells. Thus, duvelisib may target CLL B-cell growth directly, or indirectly by inhibiting the support of CD4+ T cells in the TME. The potential effect of duvelisib on the tumor-supporting myeloid compartment was also tested. Because of limited human myeloid-cell engraftment in our NSG model, we studied the effect of duvelisib on murine macrophages. Mice receiving duvelisib had reduced numbers of splenic CD11b+ GR-1low LY-6Clow LY-6Gneg macrophages compared to controls, suggesting duvelisib altered macrophage development. Prior in vitro studies demonstrated inhibition of CLL B-cell survival and proliferation by duvelisib, as well as blockade of T-cell migration and M2 macrophage polarization (Balakrishnan 2015; Peluso 2014). Our current in vivo studies further support duvelisib's effect on CLL B-cell growth and survival through inhibition of cellular homing to supportive tissue niches and alterations in the TME. The latter, in part, is through suppression of T-cell support and alterations in the macrophage compartment. Overall, these preclinical results suggest that inhibition of PI3K-δ and PI3K-γ by duvelisib affects CLL cell survival through direct and indirect mechanisms. Disclosures McGovern: Infinity Pharmaceuticals, Inc.: Employment. Kutok:Infinity Pharmaceuticals, Inc.: Employment.

Description: HMGA2 is a member of the high-mobility group A family and plays a role in the regulation of gene transcription and chromatin structure. HMGA2 is a validated target of the let-7 family of miRNAs. Let-7 miRNAs are highly regulated in erythroid cells during the fetal-to-adult developmental transition (1). Recent studies demonstrated that the LIN28 -let-7 axis mediated up-regulation of fetal hemoglobin (HbF) expression to 〉30% of the total globin levels in cultured erythroblasts from adult humans (2) and the amelioration of hypoxia-related sickling of cultured mature erythrocytes from pediatric patients with sickle cell disease (3). Interestingly, increased expression of endogenous HbF in a patient receiving gene therapy was also associated with truncated HMGA2 protein expression after lentiviral integration and disruption of let-7 targeting at the HMGA2 gene locus (4). Therefore, we hypothesized that HMGA2 may be involved in fetal hemoglobin regulation as a downstream target of the let-7 miRNAs. To study the effects of HMGA2 upon erythropoiesis and globin expression, lentiviral constructs were designed for let-7 resistant expression of HMGA2 driven by the erythroid-specific gene promoter region of the human SPTA1 gene (HMGA2 -SPTA1-OE), with a matched empty vector control. Transductions were performed in CD34+ cells from four adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Overexpression of HMGA2 was confirmedby Q-RT-PCR (control: below detection limits; HMGA2 -SPTA1-OE: 2.51E+04 ± 3.44E+04 copies/ng) and Western blot analyses at culture day 14. Cell counting revealed no significant changes between HMGA2 -SPTA1-OE and control (empty vector) transductions at culture day 14. Terminal maturation with loss of CD71 from the erythroblast cell surface and enucleation assessed by thiazole orange staining were analyzed in the control and HMGA2 -SPTA1 -OE samples at the end of the culture period. Globin genes expression levels were evaluated for HMGA2 -SPTA1-OE by Q-RT-PCR. HMGA2 -SPTA1-OE caused a significant increase in gamma-globin mRNA expression levels compared to controls (control: 5.02E+05 ± 8.62E+04 copies/ng; HMGA2 -SPTA1-OE: 1.45E+06 ± 7.31E+05 copies/ng; p=0.037). Consistent with the increase in gamma-globin mRNA levels, HPLC analyses at culture day 21 demonstrated modest but significant increases in HbF levels in HMGA2 -SPTA1-OE compared to controls (HbF control: 5.41 ± 2.15%; HMGA2 -SPTA1-OE: 16.53 ± 4.43%; p=0.006). Possible effect(s) and downstream mechanism(s) triggered by HMGA2 -SPTA1-OE were investigated. Q-RT-PCR analyses demonstrated no significant changes in the let-7 family of miRNAs in HMGA2 -SPTA1-OE compared to controls. Expression patterns of several transcription factors such as BCL11A, KLF1, SOX6 and GATA1 were investigated by Q-RT-PCR and no significant changes were detected in HMGA2 -SPTA1-OE compared to controls. While BCL11A mRNA levels were decreased by HMGA2 -SPTA1 -OE, the differences did not reach statistical significance (control: 4.26E+02 ± 8.18E+01 copies/ng; HMGA2 -SPTA1 -OE: 2.84E+02 ± 1.48E+02 copies/ng; p=0.104). However, nuclear BCL11A protein levels assessed by Western analysis were suppressed in HMGA2 -SPTA1 -OE. In summary, these results demonstrate that HMGA2, a validated target of let-7 miRNAs, causes moderately increased gamma-globin gene and protein expression in human erythroblasts, and reduces levels of BCL11A protein. These data thus support the notion that suppression of let-7 miRNAs increases fetal hemoglobin, in part, by the targeting of erythroblast HMGA2 mRNA. (1) Noh SJ et al. J Transl Med. 7:98 (2009). (2) Lee YT et al. Blood. 122:1034-41 (2013). (3) Vasconcellos JF et al. PLoS One. 9:e106924 (2014). (4) Cavazzana-Calvo M et al. Nature. 467:318-22 (2010). Disclosures No relevant conflicts of interest to declare.

Description: Key Points The G9a methyltransferase inhibitor UNC0638 increased pancellular expression of HbF to levels greater than 30% in adult human erythroblasts. UNC0638 altered globin locus epigenetic status/protein occupancy favoring LCR interaction with fetal genes at the expense of adult genes.

Description: Recent studies demonstrated that IGF2BP1 over-expression (IGF2BP1-OE) in adult erythroblasts has robust effects on fetal hemoglobin (HbF; 〉65% of the total globin levels), accompanied by reversal of the beta-like globin expression patterns to a fetal-like phenotype. Here we investigated if another member of the insulin-like growth factor 2 mRNA-binding protein family, IGF2BP3, also has potential for HbF regulation that may be useful for therapeutic application among patients with beta-hemoglobin disorders. The developmental pattern and expression levels for IGF2BP3 were initially determined in cord blood versus adult blood CD34(+) samples cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. RNA samples were collected at culture day 14 and expression levels were measured by qRT-PCR. IGF2BP3 showed a developmentally regulated expression pattern similar to IGF2BP1 (IGF2BP1: cord blood: 1.3.E+03 ± 4.3.E+02 and adult blood: below detection limits; IGF2BP3: cord blood: 5.8.E+02 ± 2.4.E+02 and adult blood: below detection limits). These results were confirmed in vivo by comparing human fetal liver to adult bone marrow samples (IGF2BP1: fetal liver: 3.5.E+02 ± 5.7.E+01, adult bone marrow: below detection limits and IGF2BP3: fetal liver: 2.0.E+01 ± 2.7.E+00, adult bone marrow: below detection limits). To investigate the effects of IGF2BP3 upon erythropoiesis and globin expression, a lentiviral construct was designed for expression of IGF2BP3 driven by the erythroid-specific gene promoter region of the human SPTA1 gene (IGF2BP3-OE), with a matched empty vector control. Transductions were performed in CD34(+) cells from four adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Over-expression of IGF2BP3 was confirmedby qRT-PCR and Western blot analyses at culture day 14. IGF2BP3-OE cells maintained their ability to differentiate and enucleate ex vivo compared to donor-matched controls. The expression levels of globin genes were evaluated at culture day 14 by qRT-PCR and showed that IGF2BP3-OE caused significantly increased gamma-globin expression levels compared to control transductions (control: 7.7.E+05 ± 1.7.E+05; IGF2BP3-OE: 8.4.E+06 ± 3.2.E+06; p=0.018). Consistent with increased gamma-globin, HbF rose to moderately high levels upon IGF2BP3-OE (control: 4.0 ± 2.1%; IGF2BP3-OE: 18.6 ± 1.0%; p=0.0021). In addition, the expression pattern of the erythroid transcription factor BCL11A was investigated by qRT-PCR at culture day 14 and no significant changes were observed (control: 5.6.E+02 ± 2.7.E+02; IGF2BP3-OE: 6.7.E+02 ± 3.5.E+02; p=0.694). However, minor decreases in BCL11A protein levels were detected by Western analysis. These results demonstrate that IGF2BP3 is developmentally regulated in human erythroid tissues with silencing during the fetal-to-adult transition. However, the effects of IGF2BP3-OE on HbF levels were less robust when compared to IGF2BP1-OE in cultured adult erythroblasts. Disclosures No relevant conflicts of interest to declare.